Synthetic mammalian α-n-acetylglucosaminidase and genetic sequences encoding same

ABSTRACT

The present invention relates generally to mammalian α-N-acetyglucosaminidase and to genetic sequences encoding same and to their use in the investigation, diagnosis and treatment of subjects suspected of or suffering from α-N-acetylglucosaminidase deficiency.

FIELD OF THE INVENTION

The present invention relates generally to mammalian α-N-acetylglucosaminidase and to genetic sequences encoding same and to the use of these in the investigation, diagnosis and treatment of subjects suspected of or suffering from α-N-acetylglucosaminidase deficiency.

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography.

Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

BACKGROUND TO THE INVENTION

The increasing sophistication of recombinant DNA technology is greatly facilitating the efficacy of many commercially important industries including areas of medical and pharmaceutical research and development. The ability to purify native proteins and subsequently clone genetic sequences encoding these proteins is an important first step in the development of a range of therapeutic and diagnostic procedures. However, practitioners have faced many difficulties in purifying target molecules to an extent sufficient to determine amino acid sequences to permit the development of oligonucleotide probes to assist in the cloning of genetic sequences encoding the target molecules.

Such difficulties have been particularly faced in the research and development of lysosomal enzymes. An important lysosomal enzyme is α-N-acetylglucosaminidase (EC 2.1.50). This enzyme acts as a exoglycosidase in lysosomes to hydrolyse the terminal α-N-acetylglucosamine residues present at the non-reducing terminus of fragments of heparan sulphate and heparin (Hopwood, 1989). A deficiency in this lysosomal hydrolase is responsible for the pathogenesis of Sanfilippo B (Mucopolysaccharidosis type IIIB [MPS-IIIB]) syndrome (von-Figura and Kresse, 1972; O’Brien, 1972). This is an autosomal recessive disorder of glycosaminoglycan catabolism leading to storage and excretion of excessive amounts of heparan sulphate and a variety of clinical phenotypes, but classically presenting with progressive mental retardation in conjunction with skeletal deformities (McKusick and Neufeld, 1983).

There is a need, therefore, to purify α-N-acetylglucosaminidase and to clone genetic sequences encoding same to permit development of a range of therapeutic and diagnostic procedures to assist in the diagnosis and treatment of disease conditions arising from α-N-acetylglucosaminidase deficiency.

SUMMARY OF THE INVENTION

One aspect of the invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a mammalian α-N-acetylglucosaminidase or fragment or derivative thereof.

A second aspect of the invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which is capable of hybridising under at least low stringency conditions to a nucleotide sequence set forth in SEQ ID NO:1 SEQ ID NO:3 or a complementary strand or a homologue, analogue or derivative thereof.

Another aspect of the invention is directed an isolated nucleic acid molecule which is at least 40% identical to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or to a complementary strand thereof or a homologue, analogue or derivative thereof.

A further aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide capable of hydrolysing the terminal α-N-acetylglucosamine residues present at the non-reducing terminus of figments of heparan sulphate and heparin residues and wherein said nucleotide sequence is capable of hybridising under low stringency conditions to the nucleotide sequence set forth in SEQ ID NO:1.

A further aspect of the invention is directed to a genetic construct comprising a sense molecule, for the expression or over-expression of α-N-acetylglucosaminidase in prokaryotic or eukaryotic cells.

A further aspect of the present invention is directed to synthetic α-N-acetylglucosaminidase or like molecule.

A further aspect of the invention contemplates antibodies to α-N-acetylglucosaminidase and preferably synthetic α-N-acetylglucosaminidase or a like molecule.

In still yet another aspect of the present invention there is contemplated a method of diagnosing a mutation or other abberations in the α-N-acetylglucosaminidase gene in a human or animal patient.

Another aspect contemplates a method of treating patients suffering from α-N-acetylglucosaminidase deficiency, such as in MPS-IIIB, said method comprising administering to said patient an effective amount of α-N-acetylglucosaminidase or active like form thereof.

Another aspect of the present invention is directed to a pharmaceutical composition comprising a recombinant mammalian α-N-acetylglucosaminidase or an active fragment or derivative thereof and one or more pharmaceutically acceptable carriers and/or diluents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photographic representation of α-N-acetylglucosaminidase purified from human placenta following SDS/PAGE. Lane 1: M_(r) standards (kDa); Lanes 2 and 3: purified α-N-acetylglucosaminidase from human placenta. Lane 4 and 5, bovine serum albumin.

FIG. 2 is a photographic representation of an SDS/polyacrylamide gel showing the molecular weights of recombinant α-N-acetylglucosaminidase polypeptides produced in CHO cells before (−) and after (+) PNGase F digestion. The 50 mM NaCl and 75 mM NaCl fractions are indicated. Molecular weights of α-N-acetylglucosaminidase polypeptides are indicated on the left of the figure. Molecular weights of marker proteins are indicated on the right hand side of the figure (lane 5).

Single and three letter abbreviations of conventional amino acid residues as used herein are defined in Table 1.

Suitable amino acid substitutions referred to herein are defined in Table 2.

Codes for non-conventional amino acid residues as used herein are defined in Table 3.

TABLE 1 Three-letter One-letter Amino Acid Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X

TABLE 2 Suitable residues for amino acid substitutions Original Residue Exemplary Subsitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

TABLE 3 Non-conventional Non-conventional amino acid Code amino acid Code α-amniobutyric acid Abu L-N-methylalanine Nmala α-amino-α- Mgabu L-N-methylarginine Nmarg methylbutyrate aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric Aib L-N-methylcysteine Nmcys acid aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-y-aminobutyrate Mgabu D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen D-α- Dmasn α-methyl-α-napthylalanine Manap methylasparagine D-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D-α- Dmgln N-(2-aminoethyl)glycine Naeg methylglutamine D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn D-α- Dmile N-amino-α-methylbutyrate Nmaabu methylisoleucine D-α-methylleucine Dmleu α-napthylalanine Anap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α- Dmmet N-(2-carbamylethyl)glycine Ngln methylmethionine D-α- Dmorn N-(carbamylmethyl)glycine Nasn methylornithine D-α- Dmphe N-(2-carboxyethyl)glycine Nglu methylphenylalanine D-αa-methylproline Dmpro N-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycine Ncbut D-α- Dmthr N-cycloheptylglycine Nchep methylthreonine D-α- Dmtrp N-cyclohexylglycine Nchex methyltryptophan D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec D-α-methylvaline Dmval N-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro D-N- Dnmasn N-cycloundecylglycine Ncund methylasparagine D-N- Dnmasp N-(2,2-diphenylethyl) methylaspartate glycine Nbhm D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl) glycine Nbhe D-N- Dnmgln N-(3-guanidinopropyi) Narg methylglutamine glycine D-N- Dnmglu N-(1-hydroxyethyl)glycine Nthr methylglutamate D-N- Dnmhis N-(hydroxyethyl))glycine Nser methylhistidine D-N- Dnmile N-(imidazolylethyl)) Nhis methylisoleucine glycine D-N-methylleucine Dnmleu N-(3-indolylyethyl) Nhtrp glycine D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate Nmgabu N-methyl- Nmchexa D-N-methylmethionine Dnmmet cyclohexylalanine D-N- Dnmorn N-methylcyclopentylalanine Nmcpen methylornithine N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methyl- Nmaib D-N-methylproline Dnmpro aminoisobutyrate N-(1-methyl- Nile D-N-methylserine Dmnser propyl)glycine N-(2-methyl- Nleu D-N-methylthreonine Dnmthr propyl)glycine D-N- Dnmtrp N-(1-methylethyl)glycine Nval methyltryptophan D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen y-aminobutyric acid Gabu N-p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen L- Hphe L-α-methylalanine Mala homophenylalanine L-α-methylarginine Marg L-α-methylasparagine Masn L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug L-α-methylcysteine Mcys L-methylethylglycine Metg L-α- Mgln L-α-methylglutamate Mglu methylglutamine L-α-methylhistidine Mhis L-α-methylhomo Mhphe phenylalanine L-α- Mile N-(2-methylthioethyl) Nmet methylisoleucine glycine L-α-methylleucine Mleu L-α-methyllysine Mlys L-α- Mmet L-α-methylnorleucine Mnle methylmethionine L-α- Mnva L-α-methylornithine Morn methylnorvaline L-α- Mphe L-α-methylproline Mpro methylphenylalanine L-α-methylserine Mser L-α-methylthreonine Mthr L-α- Mtrp L-α-methyltyrosine Mtyr methyltryptophan L-α-methylvaline Mval L-N-methythomo phenylalanine Nmhphe N-(N-(2,2- N-(N-(3,3-diphenylpropyl) diphenylethyl) carbamylmethyl) Nnbhm carbamylmethyl)glycine Nnbhe glycine 1-carboxy-1- (2,2-diphenyl- ethylamino) cyclopropane Nmbc

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes, or are complementary to a sequence which encodes, a mammalian α-N-acetylglucosaminidase or fragment or derivative thereof or its like molecule.

Preferably, the mammal is a human, livestock animal, companion animal, wild animal or laboratory test animal (e.g. rabbit, rat, mouse or guinea pig). Most preferably, the mammal is a human. Conveniently, the α-N-acetylglucosaminidase is isolatable from the liver, kidney or placenta. However, the present invention extends to all mammalian α-N-acetylglucosaminidase enzymes and from any anatomical or cellular source and/or any biological fluid source, such as but not limited to plasma, serum, cell extract or lymph fluid.

Although a preferred embodiment of the present invention contemplates the use of human α-N-acetylglucosaminidase or genomic or recombinant (e.g. cDNA) genetic sequences encoding same in the investigation, diagnosis and/or treatment of human subjects (i.e. homologous system), one skilled in the art will appreciate that the enzyme or genetic sequences encoding same from a non-human animal may also be useful. Such a heterologous system is encompassed by the present invention.

The term “nucleic acid molecule” as used herein shall be taken to refer to any RNA or DNA (e.g. cDNA) molecule, whether single-stranded or double-stranded or in a linear or covalently-closed form. The nucleic acid molecule may also be DNA corresponding to the entire genomic gene or a substantial portion thereof or a fragment or derivative thereof.

The nucleic acid molecule of the present invention may constitute solely the nucleotide sequence encoding α-N-acetylglucosaminidase or α-N-acetylglucosaminidase-like molecule or may be part of a larger nucleic acid molecule. Accordingly, the present invention extends to the isolated genomic α-N-acetylglucosaminidase gene. The non-translated sequences in a larger nucleic acid molecule may include vector, transcriptional and/or translational regulatory sequences, promoter, terminator, enhancer, replication or signal sequences or non-coding regions (eg intron sequences) of an isolated genomic gene.

Reference herein to a “gene” is to be taken in its broadest context and includes:

(i) a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5′- and 3′- untranslated sequences);

(ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) optionally comprising 5′- or 3′-untranslated sequences of the gene; or

(iii) synthetic, amplified DNA fragments or other recombinant nucleic acid molecules produced in vitro and comprising all or a part of the coding region and/or 5′- or 3′-untranslated sequences of the gene.

The term “gene” is also used to describe synthetic or fusion molecules encoding all or part of a functional product. A functional product is one which comprises a sequence of nucleotides or is complementary to a sequence of nucleotides which encodes a functional polypeptide, in particular a polypeptide having the catalytic activity of α-N-acetylglucosaminidase or a homologue, analogue or derivative thereof.

For the present purpose, “homologues” of a nucleotide sequence shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as the nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence within said sequence, of one or more nucleotide substitutions, insertions, deletions, or rearrangements.

“Analogues” of a nucleotide sequence set forth herein shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence of any non-nucleotide constituents not normally present in said isolated nucleic acid molecule, for example carbohydrates, radiochemicals including radionucleotides, reporter molecules such as, but not limited to DIG, alkaline phosphatase or horseradish peroxidase, amongst others.

“Derivatives” of a nucleotide sequence set forth herein shall be taken to refer to any isolated nucleic acid molecule which contains significant sequence similarity to said sequence or a part thereof. Generally, the nucleotide sequence of the present invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or insertions. Nucleotide insertional derivatives of the nucleotide sequence of the present invention include 5′ and 3′ terminal fusions as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues. Insertional nucleotide sequence variants are those in which one or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of said sequence, although random insertion is also possible with suitable screening of the resulting product being performed. Deletional variants are characterised by the removal of one or more nucleotides from the nucleotide sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide or nucleotide analogue inserted in its place.

Preferably, a homologue, analogue or derivative of an α-N-acetylglucosaminidase gene according to any embodiments described herein, comprises a sequence of nucleotides of at least 10 contiguous nucleotides derived from SEQ ID NO:1 or SEQ ID NO:3 or a complementary strand thereof, wherein the sequence of said homologue, analogue or derivative is at least 40% identical to SEQ ID NO:1 or SEQ ID NO:3 or a complementary strand thereof or wherein said homologue, analogue or derivative is capable of hybridising to said sequence under at least low stringency hybridisation conditions.

For the purposes of nomenclature, the nucleotide sequence set for in SEQ ID NO: 1 relates to the cDNA encoding the human α-N-acetylglucosaminidase enzyme.

The nucleotide sequence set forth in SEQ ID NO:3 relates to the genomic gene equivalent of the cDNA encoding the human liver α-N-acetylglucosaminidase enzyme. Those skilled in the art will be aware that the specific exon sequences described in SEQ ID NO:3 correspond to the coding regions of the α-N-acetylglucosaminidase gene, said exon regions further comprising the entire open reading frame of the cDNA sequence set forth in SEQ ID NO:1, when aligned in a head-to-tail configuration. The intron sequences of SEQ ID NO:3, which correspond to non-coding regions of the gene which are spliced from the primary transcription product thereof, although not explicitly defined, may be readily deduced by those skilled in the art, when provided with the exon sequence data provided in the nucleotide sequence listing.

The nucleotide sequence of the present invention may correspond to the sequence of the naturally-occurring α-N-acetylglucosaminidase gene or may comprise a homologue, analogue or derivative thereof which contains single or multiple nucleotide substitutions, deletions and/or additions. All such homologues, analogue or derivatives encode α-N-acetylglucosaminidase or α-N-acetylglucosaminidase-like molecules or a homologue, analogue or derivative thereof as contemplated by the present invention. The length of the nucleotide sequence may vary from a few bases, such as in nucleic acid probes or primers, to a full length sequence.

The present invention is particularly directed to the nucleic acid in cDNA form and particularly when inserted into an expression vector. The expression vector may be replicable in a eukaryotic or prokaryotic cell and may either produce mRNA or the mRNA may be subsequently translated into α-N-acetylglucosaminidase or like molecule. Particularly preferred eukaryotic cells include CHO cells but may be in any other suitable mammalian cells or cell lines or non-mammalian cells such as yeast or insect cells.

In an alternative embodiment, the present invention provides a nucleic acid molecule comprising a sequence of nucleotides which encodes or are complementary to a sequence which encodes a polypeptide capable of hydrolysing the α-N-acetylglucosamine residues from the non-reducing terminus of heparan sulphate and heparin fragments and wherein said nucleotide sequence is capable of hybridising under at least low stringency conditions to a nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or a homologue, analogue or derivative thereof.

A second aspect of the invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which is capable of hybridising under at least low stringency conditions to a nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or a complementary strand or a homologue, analogue or derivative thereof.

Preferably, hybridisation is possible under at least medium stringent conditions. More preferably, hybridisation is possible under high stringent conditions.

For the purposes of defining the level of stringency, reference can conveniently be made to Sambrook et al (1989) or Ausubel et al (1987) which are herein incorporated by reference.

A low stringency is defined herein as being a hybridisation and/or wash carried out in 4-6X SSC/0.1-0.5% w/v SDS at 37-45° C. for 2-3 hours. A medium stringency hybridisation and/or wash is carried out in 1-4X SSC/0.25-0.5% w/v SDS at ≧45° C. for 2-3 hours and a high stringency hybridisation and/or wash is carried out 0.1-1X SSC/0.1% w/v SDS at 60° C. for 1-3 hours.

Alternative conditions of stringency may be employed to those specifically recited herein. Generally, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash. Those skilled in the art will be aware that the conditions for hybridisation and/or wash may vary depending upon the nature of the hybridisation membrane or the type of hybridisation probe used. Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of clarification of parameters affecting hybridisation between nucleic acid molecules, reference is found in pages 2.10.8 to 2.10.16. of Ausubel et al. (1987), which is herein incorporated by reference.

Those skilled in the art will be aware that the nucleotide sequences set forth in SEQ ID NO:1 and SEQ ID NO:3 may be used to isolate the corresponding genes from other human tissues or alternatively, from the tissues or cells of other species, without undue experimentation. Means for the isolated of such related sequences will also be known to those skilled in the art, for example nucleic acid hybridisation, polymerase chain reaction, antibody screening of expression libraries, functional screening of expression libraries, or complementation of mutants, amongst others. The present invention is not to be limited by the source from which the specific gene sequences described herein have been isolated or by the means used to isolate said sequences.

In one embodiment, a related genetic sequence comprising genomic DNA, or mRNA, or cDNA is contacted with a hybridisation effective amount of a genetic sequence which encodes α-N-acetylglucosaminidase, or its complementary nucleotide sequence or a homologue, analogue, derivative or functional part thereof, and then said hybridisation is detected using a suitable detection means.

The related genetic sequence may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell. Preferably, the related genetic sequence originates from an animal species or a human. More preferably, the related genetic sequence originates from a human.

Preferably, the genetic sequence which encodes α-N-acetylglucosaminidase (i.e probe or later genetic sequence) comprises a sequence of nucleotides of at least 10 nucleotides, more preferably at least 20 nucleotides, even more preferably at least 50 nucleotides and even still more preferably at least 100 nucleotides derived from the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:3 or a complementary sequence or a homologue, analogue or derivative thereof.

Preferably, the detection means is a reporter molecule capable of giving an identifiable signal (e.g. a radioisotope such as ³²P or ³⁵S or a biotinylated molecule) covalently attached to the α-N-acetylglucosaminidase probe.

In an alternative embodiment, the detection means is a polymerase chain reaction. According to this embodiment, two opposing non-complementary nucleic acid “primer molecules” of at least 10 nucleotides in length, more preferably at least 20 nucleotides in length, derived from the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 may be contacted with a nucleic acid “template molecule” and specific nucleic acid molecule copies of the template molecule amplified in a polymerase chain reaction.

The opposing primer molecules are selected such that they are each capable of hybridising to complementary strands of the same template molecule, wherein DNA polymerase-dependant DNA synthesis occurring from a first opposing primer molecule will be in a direction toward the second opposing primer molecule.

Accordingly, both primers hybridise to said template molecule such that, in the presence of a DNA polymerase enzyme, a cofactor and appropriate substrate, DNA synthesis occurs in the 5′ to 3′ direction from each primer molecule towards the position on the DNA where the other primer molecule is hybridised, thereby amplifying the intervening DNA.

Those skilled in the art are aware of the technical requirements of the polymerase chain reaction and are capable of any modifications which may be made to the reaction conditions. For example, of the polymerase chain reaction may be used in any suitable format, such as amplified fragment length polymorphism (AFLP), single-strand chain polymorphism (SSCP), amplification and mismatch detection (AMD), interspersed repetitive sequence polymerase chain reaction (IRS-PCR), inverse polymerase chain reaction (iPCR) and reverse transcription polymerase chain reaction (RT-PCR), amongst others, to isolate a related α-N-acetylglucosaminidase gene sequence or identify a mutation in an α-N-acetylglucosaminidase genetic sequence. Such variations of the polymerase chain reaction are discussed in detail by McPherson et al (1991), which is incorporated herein by reference. The present invention encompasses all such variations, the only requirement being that the final product of the reaction is an isolated nucleic acid molecule which is capable of encoding α-N-acetylglucosaminidase or a homologue, analogue or derivative thereof.

In a preferred embodiment, the first primer molecule is preferably derived from the sense strand of a gene which encodes α-N-acetylglucosaminidase, in particular from the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or a homologue, derivative or analogue thereof and the second primer molecule is preferably derived from the antisense strand of said gene.

Those skilled in the art will be aware that it is not essential to the performance of the invention that the primer molecules be derived from the same gene.

According to this embodiment, the nucleic acid primer molecule may further consist of a combination of any of the nucleotides adenine, cytidine, guanine, thymidine, or inosine, or functional analogues or derivatives thereof, capable of being incorporated into a polynucleotide molecule provided that it is capable of hybridising under at least low stringency conditions to the nucleic acid molecule set forth in SEQ ID NO:1 or SEQ ID NO:3 or a homologue, analogue or derivative thereof.

The nucleic acid primer molecules may further be each contained in an aqueous pool comprising other nucleic acid primer molecules. More preferably, the nucleic acid primer molecule is in a substantially pure form.

The nucleic acid template molecule may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell. Preferably, the related genetic sequence originates from a cell, tissue, or organ derived from an animal species or a human. More preferably, the related genetic sequence originates from a cell, tissue, or organ derived from a human.

Accordingly, a third aspect of the present invention extends to an isolated nucleic acid molecule which is at least 40% identical to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or to a complementary strand thereof or a homologue, analogue or derivative thereof.

Preferably, the percentage identity to SEQ ID NO:1 or SEQ ID NO:3 is at least about 55%, still more preferably at least about 65%, yet still more preferably at least about 75-80% and even still more preferably at least about 85-95%.

In an even more preferred embodiment, the present invention provides an isolated nucleic acid molecule which is at least 40% identical to the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or to a complementary stand thereof or a homologue, analogue or derive thereof and is capable of hybridising under at least low stringency conditions to a nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3.

In a particularly preferred embodiment, the isolated nucleic acid molecule described herein is further capable of encoding a sequence of amino acids which is capable of carrying out the enzyme reaction catalysed by a α-N-acetylglucosaminidase enzyme.

The isolated nucleic acid molecule of the present invention is also useful for developing a genetic construct comprising a sense molecule, for the expression or over-expression of α-N-acetylglucosaminidase in prokaryotic or eukaryotic cells. Particularly preferred eukaryotic cells include CHO cells but may be in any other suitable mammalian cells or cell lines or non-mammalian cells such as yeast or insect cells.

The term “sense molecule” as used herein shall be taken to refer to an isolated nucleic acid molecule of the invention as described herein, which is provided in a format suitable for its expression to produce a recombinant polypeptide, when said sense molecule is introduced into a host cell.

In a particularly preferred embodiment, a sense molecule which encodes the α-N-acetylglucosaminidase comprises a sequence of nucleotides set forth in SEQ ID NO:1 or SEQ ID NO:3 or a complementary strand, homologue, analogue or derivative thereof.

In a most particularly preferred embodiment, the sense molecule of the invention comprises the sequence of nucleotides set forth in SEQ ID NO:1 or a complementary strand, homologue, analogue or derivative thereof.

Those skilled in the art will be aware that expression of a sense molecule may require the nucleic acid molecule of the invention to be placed in operable connection with a promoter sequence to produce a “sense construct”. The choice of promoter for the present purpose may vary depending upon the level of expression of the sense molecule required and/or the tissue-specificity or developmental-specificity of expression of the sense molecule which is required. The sense construct may further comprise a terminator sequence and be introduced into a suitable host cell where it is capable of being expressed to produce a recombinant polypeptide gene product.

In the context of the present invention, a sense molecule which corresponds to a genetic sequence or isolated nucleic acid molecule which encodes α-N-acetylglucosaminidase polypeptide or a homologue, analogue or derivative thereof, placed operably under the control of a suitable promoter sequence, is introduced into a cell using any suitable method for the transformation of said cell and said genetic sequence or isolated nucleic acid molecule is expressed therein to produce said polypeptide.

The present invention clearly extends to genetic constructs designed to facilitate expression of any nucleic acid molecule described herein.

A genetic construct of the present invention comprises the foregoing sense molecule, placed operably under the control of a promoter sequence capable of regulating the expression of the said nucleic acid molecule in a prokaryotic or eukaryotic cell, preferably a mammalian cell such as a CHO cell, a yeast cell, insect cell or bacterial cell. The said genetic construct optionally comprises, in addition to a promoter and sense molecule, a terminator sequence.

The term “terminator” refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3′-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3′-end of a primary transcript. Terminators active in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants.

Reference herein to a “promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. A promoter is usually, but not necessarily, positioned upstream or 5′, of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.

In the present context, the term “promoter” is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of said sense molecule in a cell.

Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of the sense molecule and/or to alter the spatial expression and/or temporal expression of said sense molecule. For example, regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence driving expression of a sense molecule, thereby conferring copper inducibility on the expression of said molecule.

Placing a sense molecule under the regulatory control of a promoter sequence means positioning the said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5′ (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.

Examples of promoters suitable for use in genetic constructs of the present invention include viral, fungal, bacterial, animal and plant derived promoters capable of functioning in animal, human, yeast, insect or bacterial cells. The promoter may regulate the expression of the said molecule constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to steal stimuli such as physiological stresses, or plant pathogens, or metal ions, amongst others. Preferably, the promoter is capable of regulating expression of a sense molecule in a cell derived from an animal species or human.

In a particularly preferred embodiment, the promoter is derived from the genomic gene encoding α-N-acetylglucosaminidase, preferably the human α-N-acetylglucosaminidase gene. In a more preferred embodiment, however, the promoter is derived from the nucleotide sequence set forth in SEQ ID NO:3 or is at least capable of hybridising to nucleotide residues 1 to 989 of SEQ ID NO:3 or at least 20 contiguous nucleotides derived therefrom.

In an even more particularly preferred embodiment, the promoter is the CMV promoter sequence or a promoter sequence derived therefrom.

An alternative embodiment of the invention is directed to a genetic construct comprising a promoter or functional derivative, part fragment, homologue, or analogue thereof, derived from the α-N-acetylglucosaminidase genomic gene defined by SEQ ID NO:3.

Preferably, said genetic construct further comprises the α-N-acetylglucosaminidase sequence defined by SEQ ID NO:1 placed in operably connection with said promoter.

A further aspect of the present invention is directed to synthetic α-N-acetylglucosaminidase or like molecule.

The term “synthetic” as used herein shall be taken to include both recombinant and chemically-synthesised molecules produced by the sequential addition of amino acid residues or groups of amino acid residues in defined order.

In one embodiment, the invention relates to recombinant α-N-acetylglucosaminidase or like molecule encoded by or expressed from the nucleic acid molecules as hereinbefore described.

In another embodiment, the synthetic α-N-acetylglucosaminidase or like molecule comprises a sequence of amino acids which is at least 40% identical to the amino acid sequence set forth in any one of SEQ ID Nos:2, 4, 5 or 6.

More preferably, the percentage identity is at least 60% and still more preferably at least 80% or 85-90%.

A particularly preferred embodiment of the present invention provides a synthetic α-N-acetylglucosaminidase as hereinbefore defined which comprises a sequence of amino acids substantially as set forth in any one of SEQ ID Nos:2, 4, 5 or 6 or a homologue, analogue or derivative thereof.

For the purposes of nomenclature, the amino acid sequence set forth in SEQ ID NO:2 comprises the full-length translation product of the human α-N-acetylglucosaminidase gene (i.e. hereinafter referred to as the “α-N-acetylglucosaminidase polypeptide” or “SEQ ID NO:2”) produced by expression of either the cDNA sequence defined by SEQ ID NO:1 or the genomic gene defined by SEQ ID NO:3. The α-N-acetylglucosaminidase polypeptide comprises at least seven potentially-glycosylated Asn residues, at positions 261, 272, 435, 503, 513, 526 and 532. Furthermore, the amino acid sequence of the α-N-acetylglucosaminidase polypeptide may comprise a signal peptide of approximately 23 amino acid residues in length, with a probable site for signal peptide peptidase cleavage occurring between Gly₂₃ and Asp₂₄.

The amino acid sequences set forth in SEQ ID Nos:4-6 relate to N-terminal and internal (i.e. CNBr) amino acid sequences derived from human α-N-acetylglucosaminidase, purified as described in Example 1. As described in Example 2, the purified form of the enzyme comprises two polypeptides having approximate molecular weights of 82 and 77 kDa. The sequence set forth in SEQ ID NO:4 relates to the N-terminal sequence of the 82 kDa polypeptide, while SEQ ID NO:5 relates to the N-terminal sequence of the 77 kDa polypeptide. Furthermore, SEQ ID NO:4 comprises amino acids residues 24-43 of SEQ ID NO:2, while SEQ ID NO:5 comprises amino acid residues 59-76 of SEQ ID NO:2.

The amino acid sequence defined by SEQ ID NO:6 relates to the CNBr-cleaved peptide of purified human α-N-acetylglucosaminidase. This amino acid sequence aligns with amino acid residues 540-554 of the α-N-acetylglucosaminidase polypeptide (SEQ ID NO:2).

In the present context, “homologues” of a polypeptide refer to those polypeptides, enzymes or proteins which have a similar α-N-acetylglucosaminidase enzyme activity, notwithstanding any amino acid substitutions, additions or deletions thereto. A homologue may be isolated or derived from the same or another animal species.

Furthermore, the amino acids of a homologous polypeptide may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, charge or antigenicity, and so on.

“Analogues” encompass α-N-acetylglucosaminidase polypeptides and peptide derivatives thereof notwithstanding the occurrence of any non-naturally occurring amino acid analogues therein.

The term “derivative” in relation to the polypeptides of the invention refer to mutants, parts or fragments of a functional molecule. Derivatives include modified peptides in which ligands are attached to one or more of the amino acid residues contained therein, such as carbohydrates, enzymes, proteins, polypeptides or reporter molecules such as radionuclides or fluorescent compounds. Glycosylated, fluorescent, acylated or alkylated forms of the subject peptides are particularly contemplated by the present invention. Additionally, derivatives of a polypeptide may comprise fragments or parts of an amino acid sequence disclosed herein and are within the scope of the invention, as are homopolymers or heteropolymers comprising two or more copies of the subject polypeptides. Procedures for derivatizing peptides are well-known in the art.

Accordingly, this aspect of the present invention is directed to any proteinaceous molecule comprising an amino acid sequence corresponding to the full length mammalian α-N-acetylglucosaminidase enzyme or to a like molecule. The like molecule, therefore, comprises parts, derivatives and/or portions of the α-N-acetylglucosaminidase enzyme whether functional or not.

Preferably, the mammal is human but may be of non-human origin as contemplated above.

The synthetic or recombinant α-N-acetylglucosaminidase of the present invention may comprise an amino acid sequence corresponding to the naturally occurring amino acid sequence or may contain single or multiple amino acid substitutions, deletions and/or additions. The length of the amino acid sequence may range from a few residues to a full length molecule.

Amino acid substitutions are typically of single residues. Amino acid insertions will usually be in the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues. Preferably, deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.

Amino acid insertional derivatives of α-N-acetylglucosaminidase of the present invention include amino and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Typical substitutions are those made in accordance with the following Table 2:

The amino acid variants referred to above may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis (Merrifield synthesis) and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in DNA having known or partially known sequence are well known and include, for example, M13 mutagenesis. The manipulation of DNA sequence to produce variant proteins which manifest as substitutional, insertional or deletional variants are conveniently elsewhere described such as Sambrook et al, 1989 Molecular Cloning: A Laboratory Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.

The derivatives or like molecules include single or multiple substitutions, deletions and/or additions of any component(s) naturally or artificially associated with the α-N-acetylglucosaminidase enzyme such as carbohydrate, lipid and/or other proteinaceous moieties. For example, the present invention extends to glycosylated and non-glycosylated forms of the molecule. All such molecules are encompassed by the expression “mutants”, “derivatives”, “fragments”, “portions” and “like” molecules. These molecules may be active or non-active and may contain specific regions, such as a catalytic region. Particularly, preferred derivative molecules include those with altered glycosylation patterns relative to the naturally occurring molecule. Even more particularly, the recombinant molecule is more highly glycosylated than the naturally occurring molecule. Such highly glycosylated derivatives may have improved take-up properties and enhanced half-lives.

As indicated in the Examples, the molecular weight of purified human α-N-acetylglucosaminidase (i.e. 82 kDa and 77 kDa) and recombinant mammalian α-N-acetylglucosaminidase produced in CHO cells (i.e. 89 kDa and 79 kDa) are greater than the deduced molecular weight of the α-N-acetylglucosaminidase polypeptide set forth in SEQ ID No:2 (i.e. 70 kDa), suggesting that the purified and recombinant polypeptide are post-translationally modified. The data presented in Example 8 indicate further that the recombinant α-N-acetylglucosaminidase enzyme produced in CHO cells, at least, is glycosylated and that the difference in molecular weight determined for the recombinant polypeptides and the polypeptide of SEQ ID) No:2 is due almost entirely to glycosylation of the recombinant polypeptide by CHO cells. As shown in Example 9, the glycosylated recombinant α-N-acetylglucosaminidase polypeptide exhibits enzymatic activity.

The present invention also extends to synthetic α-N-acetylglucosaminidase or like molecules when fused to other proteinaceous molecules. The latter may include another enzyme, reporter molecule, purification site or an amino acid sequence which facilitates transport of the molecule out of a cell, such as a signal sequence.

The present invention extends further to post-translational modifications to the α-N-acetylglucosaminidase enzyme. The modifications may be made to the naturally occurring enzyme or following synthesis by recombinant techniques. The modifications may be at the structural level or at, for example, the electrochemical level such as modifying net charge or structural conformation of the enzyme.

Such modification may be important to facilitate entry or penetration of the enzyme into selected issues such as cartilage or blood brain barriers or to increase circulation half-life.

Analogues of α-N-acetylglucosaminidase contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the enzyme.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄; amidination with methylacetinidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5′-phosphate followed by reduction with NaBH₄.

The guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenyisulphonic acid, phenylmercury chloride, 2-chloromercuric-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alklylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, omithine, sarcosine, 4amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. Non-naturally occurring amino acids contemplated by the present invention are incorporated herein, as Table 3.

Crosslinkers can be used, for example, to stabilise 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, the enzyme could be conformationally constrained by, for example, incorporation of C_(α)and N_(α)-methylamino acids, introduction of double bonds between C_(α) and C_(ρ) atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

Electrochemical modifications of α-N-acetylglucosaminidase include interaction with polylysine or polyethylene glycol or other agent which effects an overall change to the net charge of the enzyme.

Advantageously, the recombinant α-N-acetylglucosaminidase is a biologically pure preparation meaning that it has undergone some purification away for other proteins and/or non-proteinaceous material. The purity of the preparation may be represented as at least 40% of the enzyme, preferably at least 60%, more preferably at least 75%, even more preferably at least 85% and still more preferably at least 95% relative to non-α-N-acetylglucosaminidase material as determined by weight, activity, amino acid homology or similarity, antibody reactivity or other convenient means.

Particularly preferred methods for the preparation and purification of recombinant α-N-acetylglucosaminidase are provided in Examples 7 and 8.

Those skilled in the art will be aware of the means of purifying a synthetic or recombinant α-N-acetylglucosaminidase from several sources without undue experimentation and for expressing the degree of purity of such a purified preparation of the enzyme.

The present invention further contemplates antibodies to α-N-acetylglucosaminidase and preferably synthetic α-N-acetylglucosaminidase or like molecule. The antibodies may be polyclonal or monoclonal, naturally occurring or synthetic (including recombinant, fragment or fusion forms). Such antibodies will be useful in developing immunoassays for α-N-acetylglucosaminidase and for identifying additional genetic sequences which are capable of expressing α-N-acetylglucosaminidase polypeptides or homologues, analogues or derivatives thereof.

Both polyclonal and monoclonal antibodies are obtainable by immunisation with an appropriate synthetic or recombinant gene product, or epitope, or peptide fragment of a gene product, in particular a α-N-acetylglucosaminidase polypeptide or a homologue, analogue or derivative thereof.

Alternatively, fragments of antibodies may be used, such as Fab fragments. The present invention extends further to encompass recombinant and synthetic antibodies and to antibody hybrids. A “synthetic antibody” is considered herein to include fragments and hybrids of antibodies.

A further aspect of the present invention contemplates a method of screening for mutations or other abberations in the α-N-acetylglucosaminidase gene in a human or animal patient. Such a method may be accomplished in a number of ways including isolating a source of DNA to be tested or mRNA therefrom and hybridising thereto a nucleic acid molecule as hereinbefore described. Generally, the nucleic acid is probe or primer size and polymerase chain reaction is a convenient means by which to analyse the RNA or DNA. Other suitable assays include the ligation chain reaction and the strand displacement amplification methods. The α-N-acetylglucosaminidase sequence can also be determined and compared to the naturally occurring sequence. Such methods may be useful in adults and children and may be adapted for a pre-natal test The DNA to be tested includes a genomic sample carrying the α-N-acetylglucosaminidase gene, a cDNA clone and/or amplification product

In accordance with this aspect of the present invention there is provided a method for screening for abberations in the α-N-acetylglucosaminidase gene including the absence of such a gene or a portion or a substantial portion thereof comprising isolating a sample of DNA or mRNA corresponding to a region of said DNA and contacting same with an oligonucleotide probe capable of hybridising to one or more complementary sequences within the α-N-acetylglucosaminidase gene and then detecting the hybridisation, the extent of hybridisation or the absence of hybridisation.

Alternatively, the probe is a primer and capable of directing amplification of one or more regions of said α-N-acetylglucosaminidase gene and the amplification products and/or profile of amplification products is compared to an individual carrying the full gene or to a reference date base.

Conveniently, the amplification products are sequenced to determine the presence or absence of the full gene.

The present invention extends to the use of any and all DNA-based or nucleic acid-based hybridisation and/or polymerase chain reaction formats as described herein, for the diagnosis of a disorder involving the α-N-acetylglucosaminidase gene in a human or animal patient.

The present invention further ends to a method of treating patients suffering from α-N-acetylglucosaminidase deficiency, such as in MPS-IIIB, said method comprising administering to said patient an effective amount of α-N-acetylglucosaminidase or active like form thereof.

Preferably, the α-N-acetylglucosaminidase is in recombinant form. Such a method is referred to as “enzyme therapy”. Alternatively, gene therapy can be employed including introducing an active gene (i.e. a nucleic acid molecule as hereinbefore described) or to parts of the gene or other sequences which facilitate expression of a naturally occurring α-N-acetylglucosaminidase gene.

Administration of α-N-acetylglucosaminidase for enzyme therapy may be by oral, intravenous, suppository, intraperitoneal, intramuscular, intranasal, intradermal or subcutaneous administration or by infusion or implantation. The α-N-acetylglucosaminidase is preferably as hereinbefore described including active mutants or derivatives thereof and glycosylation variants thereof. Administration may also be by way of gene therapy including expression of the gene by inclusion of the gene in viral vectors which are introduced into the animal (e.g. human) host to be treated. Alternatively, the gene may be expressed in a bacterial host which is then introduced and becomes part of the bacterial flora in the animal to be tested.

Still yet another aspect of the present invention is directed to a pharmaceutical composition comprising synthetic (e.g. recombinant) α-N-acetylglucosaminidase or like molecule, including active derivatives and fragments thereof, alone or in combination with other active molecules. Such other molecules may act synergistically with the enzyme or facilitates its entry to a target cell. The composition will also contain one or more pharmaceutically acceptable carriers and/or diluents. The composition may alternatively comprise a genetic component useful in gene therapy.

The active ingredients of the pharmaceutical composition comprising the synthetic or recombinant α-N-acetylglucosaminidase or mutants or fragments or derivatives thereof are contemplated to exhibit excellent activity in treating patients with a deficiency in the enzyme when administered in an amount which depends on the particular case. The variation depends, for example, on the patient and the α-N-acetylglucosaminidase used. For example, from about 0.5 ug to about 20 mg of enzyme per animal body or, depending on the animal and other factors, per kilogram of body weight may be administered. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or in other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. Accordingly, alternative dosages in the order of 1.0 μg to 15 mg 2.0 μg to 10 mg or 10 μg to 5mg may be administered in a single or as part of multiple doses. The active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal intradermal or suppository routes or implanting (eg using slow release molecules). Depending on the route of administration, the active ingredients which comprise a synthetic (e.g. recombinant) α-N-acetylglucosaminidase or fragments, derivatives or mutants thereof may be required to be coated in a material to protect same from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. For example, the low lipophilicity of α-N-acetylglucosaminidase will allow it to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer the vaccine by other than parenteral administration, the enzyme will be coated by, or administered with, a material to prevent its inactivation. For example, the enzyme may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Conveniently, the adjuvant is Freund's Complete or Incomplete Adjuvant. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes.

The active compound may also be administered in dispersions prepared in glycerol, liquid polyethylene glycols, and/or mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient(s) into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the α-N-acetylglucosaminidase of the present invention is suitably protected as described above, the composition may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit The amount of active compound in the vaccine compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared, so that an oral dosage unit form contains between about 0.5 ug and 20 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release reparations and formulations.

As used herein “pharmaceutically acceptable carriers and/or diluents” include any and all solvents, dispersion media, aqueous solutions, comings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The present invention further relates to the use of α-N-acetylglucosaminidase or active fragment, mutant or derivative thereof in the manufacture of a medicament for the treatment of patients suffering from a deficiency in the naturally occurring enzyme (e.g. MPS-IIIB).

The present invention is further described with reference to the following non-limiting Examples.

EXAMPLE 1 Purification of α-N-Acetylglucosaminidase

α-N-acetylglucosaminidase was purified according to the method described in Weber et al. (1996). Enzyme was purified to homogeneity from human placenta. Evidence of purity is shown following SDS/PAGE which is represented in FIG. 1. All samples were reduced with dithiothreitol prior to electrophoresis.

EXAMPLE 2 Characterisation of α-N-Acetylglucosaminidase

Results presented in FIG. 1 show two polypeptides of about 82 kDa and 77 kDa molecular weight, which correspond to α-N-acetylglucosaminidase polypeptides purified from human placenta according to Example 1.

EXAMPLE 3 Amino Add Sequence Determination

The N-terminal amino acid sequences the 77 kDA and 82 kDa α-N-acetylglucosaminidase polypeptides, in addition to the amino acid sequence of an internal CNBr cleavage-product of these peptides, were determined wing the methods of Weber et al. (1996).

The amino acid sequences are shown in Table 4.

TABLE 4 N-Terminal amino add sequences (SEQ ED NO:4 and SEQ ID No: 5) and CNBr peptide sequence (SEQ ID No:6) determined from purified human α-N-Acetylglucosaminidase

TABLE 4 N-Terminal amino acid sequences (SEQ ID NO:4 and SEQ ID No:5) and CNBr peptide sequence (SEQ ID No:6) determined from purified human α-N-Acetylglucosaminidase polypeptide 82 kDa DEAREAAAVRALVARLLGPG polypeptide 77 kDa KPGLDTYSLGGGGAAX¹ VR CNBr peptide WRLLLTSAPSLX¹ TX¹ P X¹no residue could be identified for this position, indicating that this residue could be phosphorylated or glycosylated.

X¹ no residue could be identified for this postion, indicating that this residue could be phosphorylated or glycosylated.

EXAMPLE 4 Cloning of α-N-Acetylglucosaminidase cDNA

Oligonucleotide probes were prepared based on the partial amino acid sequences obtained for the purified α-N-acetylglucosaminidase polypeptides (Example 3). The probes were subsequently used to screen a human peripheral blood leukocyte cDNA library. An approximately 2.6 kbp cDNA clone was isolated encoding most of the sequence of human α-N-acetylglucosaminidase (SEQ ID NO:1).

The remaining α-N-acetylglucosaminidase coding sequence was obtained from the nucleotide sequence of the corresponding genomic gene (SEQ ID NO:3), isolated by hybridisation to a human chromosome 17 library (Weber et.al. 1996).

The complete open reading frame is 2232 nucleotides long and encodes a 743 (plus stop codon) amino acid protein. The predicted molecular mass of the longest mature protein (minus the 23 amino acid N-terminal signal peptide) is about 79,622 daltons.

The amino acid sequence of α-N-acetyglucosaminidase is shown in SEQ ID NO:2. The deduced molecular weight of the desired amino acid sequence of α-N-acetylglucosaminidase is approximately 70kDa. The probable site of signal peptide peptidase cleavage is between amino acids 23 and 24. There are seven potential N-glycosylation sites in the sequence.

The nucleotide sequence of the corresponding α-N-acetylglucosaminidase genomic gene (SEQ ID No:3) comprises 10380 bp including 889 bp of 5′ upstream sequence corresponding to at least at part of the α-N-acetylglucosaminidase promoter sequence, in addition to the nucleotide sequences of introns I, II, II, IV, V, in addition to 1326 bp of 3′-untranslated sequence.

EXAMPLE 5 Construction of an expression vector comprising the αN-Acetylglucosaminidase cDNA sequence

The cDNA insert of λ clone pbl 33, containing bases 107 to 2575 of the α-N-acetylglucosaminidase cDNA was excised with EcoRI and subcloned into pBluescript II SK-(Stratagene). A 178 bp XmnaI fragment (bases 1 to 178 of the α-N-acetylglucosaminidase cDNA) from cosmid sub-clone 6.3, containing the start codon, was cloned into the pBluescript subclone to produce a full-length cDNA sequence in addition to 101 bp of 5′ non-translated sequence as well as 245 bp of 3′ non-translated region including the polyadenylation-site, the polyA-tail and linkerDNA The full length cDNA was directionally cloned into the pCDNA3 expressionvector (Invitrogen) via the EcoRI and BawnHI sites.

EXAMPLE 6 Expression of Recombinant α-N-Acetylglucosaminidase

Chinese Hamster Ovary (CHO) cells were transfected with expressionvector using the DOTAP transfection reagent (Boehringer Mannheim) according to the manufacturers instructions. Cells were grown in Ham's F12 medium, 10% (v/v) fetal calf serum, penicillin and streptomycin sulfate at 100 μg/ml each. Cells were grown in nonselective medium for 48 h and then incubated in medium containing 750 μg/ml G418 sulfate (Geniticin) until resistant colonies emerged.

Single cell clones were grown up and 26 of them were tested for expression of recombinant α-N-acetylglucosaminidase with a fluorogenic α-N-acetylglucosaminidase substrate. (i.e. N-acetylglucosamine α-linked to 4-methylumbelliferone)

EXAMPLE 7 Large Scale α-N-Acetylglucosaminidase Production

2 g of Cytodex 2 microcarrier beads were swollen in 250 ml of PBS for 3 h at 37° C. with three changes of PBS and then autoclaved for 15 min at 120° C. (wet cycle). The beads were then rinsed with sterile growth medium (Coons/DMEM, 10% v/v fetal calf serum, penicillin and streptomycin sulfate at 100 μg/ml each and 0.1% w/v Pluronic F68) and transferred into a Techne stirrer culture flask. The microcarrier beads were inoculated with seven confluent 175 flasks of the cell clone showing the highest expression of recombinant α-N-acetylglucosaminidase. Growth medium was added up to 200 ml and the culture incubated with a stirrer speed of 20 rpm to achieve an even distribution of cells on the beads. The cells were allowed to attach to the beads for 16 h at low speed then medium was added up to 500 ml and the stirrer speed increased to 30 rpm. After a growth phase of 48 to 72 h with daily aerating to allow gas exchange the beads were completely covered with cells and the medium was exchanged for production medium (Coons/DMEM, no fetal calf serum, penicillin and streptomycin sulfate at 100 μg/ml each, 0.1% w/v Pluronic F68 and 5 mM NH₄Cl). The glucose concentration was monitored daily and the medium replaced, when glucose fell below 5 mM every 203 days. The harvested medium contained approximately 2 mg α-N-acetylglucosaminidase protein per dm³ of production medium.

EXAMPLE 8 Purification of Recombinant α-N-Acetylglucosaminidase

Production medium was dialysed against 50 mM NaAc pH 5.5 and loaded onto a heparin-agarose column equilibrated in the same buffer. After washing with NaAc buffer and NaAc/50 mM NaCl the column was eluted with 75 mM NaCl in NaAc buffer. The eluate was dialysed against 20 mM Tris/HCI pH 7.5, loaded onto a DEAE Scphacel column, washed with 25 mM NaCl in 20 mM Tris/HCl and then eluted with 50 and 75 mM NaCl in 20 mM Tris/HCl respectively.

SDS-PAGE of the two eluates showed two polypeptide bands associated with enzyme activity with apparent molecular weights of 79 and 89 kDa. The smaller α-N-acetylglucosaminidase was eluted predominantly in the 50 mM NaCI fraction whereas the 89 kDa α-N-acetylglucosaminidase polypeptide was enriched in the 75 mM NaCl fraction (FIG. 2).

The difference in apparent molecular weight of the recombinant α-N-acetylglucosaminidase polypeptides is due to the presence of additional carbohydrate side chains, since a digest with PNGase F, which cleaves off N-glycosylation groups, reduced both the 79 kDa and 89 kDa polypeptides to the polypeptide band having an apparent molecular weight of about 70 kDa (FIG. 2), which corresponds to the approximate molecular weight deduced from primary amino acid sequence data (SEQ ID No:2).

EXAMPLE 9 Characteristics of Recombinant α-N-Acetylglucosaminidase

No differences were observed between the enzyme activities of the 79 and 89 kDa recombinant α-N-acetylglucosaminidase polypeptides produced in CHO cells according to Example 7 and 8. With the fluorogenic N-acetylglucosamine α-linked to 4-methylumbelliferone (4-MU) substrate, the enzyme has a pH-optimum of 4.6 with a k_(M) of 5.34 mM and a V_(max) of 3.97×10⁶ pmol/min/mg. Towards a ³H-labelled disaccharide substrate it should a pH-optimum of 4.1 with a k_(M) of 0.0166 mM and a V_(max) of 4.48×10⁴ pmol/min/mg.

EXAMPLE 10 Mutational Analysis of Sanfilippo B Patients

Genomic DNA is isolated from cultivated skin fibroblasts of patients by extraction with Phenol/Chloroform and used to amplify the eight exons and adjacent intronic sequences individually by PCR

Primer sequences used in the amplification reaction are readily determined from the nucleotide sequences of the α-N-acetylglucosaminidase cDNA and genomic clones set forth in SEQ ID No:1 or SEQ ID No:3. Amplification conditions are also readily determined without undue experimentation. Procedures for the design of PCR primers and amplification conditions are described in detail, for example, by McPherson et al. (1991). Differences in the primary sequence can be identified by separating the PCR products on a polyacrylamide gel under non-denaturing conditions (SSCP gels). Base changes, insertions and deletions will lead to a different band pattern compared with the wildtype in most of the cases, which can be visualised either by autoradiography of the gel after labelling the DNA during the PCR or by staining unlabelled DNA in the gel with silver. PCR products which show a different band pattern are sequences to identify the change. Other patient samples can be tested for mutations and polymorphism that were found by hybridisation with wildtype—and mutation-specific oligonucleotides (ASO).

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

REFERENCES:

1. Ausubel, F. M., Brent, R, Kinton, R E, Moore, D. D., Seidman, J. G., Smith, J. A, and Struhl, K. (1987). In: Current Protocols in Molecular Biology. Wiley Interscience (ISBN 047150338).

2. Hopwood J J (1989) In: “Heparin: Chemical and Biological Properties, Clinical Applications” (Lane D W and Lindahl U, eds.), 190-229, Edward Arnold, London.

3. McKusick V and Neufeld E (1983) In: “The Metabolic Basis of Inherited Disease” (Stanbury J B, Wyngaarden J B, Fredrickson D S, Goldstein J L and Brown M S, eds), 5th Ed., 751-771, McGraw-Hill, New York.

4. McPherson, M. J., Quirke, P. and Taylor, G. R, (1991) In: PCR A Practical Approach. Oxford University Press, Oxford. (ISBN 0-19-96322L-X).

5. O’Brien J S, (1972) Proc. Natl. Acad Sci. USA 69: 1720-1722.

6. Sambrook, J., Fritsch, E., and Maniatis, T. (1989) In: “Molecular Cloning” a laboratory manual, Cold Spring Harbour.

7. Von Figura, K, and Kresse H (1972) Biochem Biophys. Res. Commun, 48: 262-269

8. Weber B, Scott H. Blanch L, Clements P, Morris C P, Anson D, Hopwood J, (1996) Nature Genetics (submitted)

6 2575 base pairs nucleic acid single linear cDNA Homo sapiens Peripheral Blood Leukocyte CDS 102..2330 1 CCCGGGCTTA GCCTTCGGGT CCACGTGGCC GGAGGCCGGC AGCTGATTGG ACGCGGGCCG 60 CCCCACCCCC TGGCCGTCGC GGGACCCGCA GGACTGAGAC C ATG GAG GCG GTG 113 Met Glu Ala Val 1 GCG GTG GCC GCG GCG GTG GGG GTC CTT CTC CTG GCC GGG GCC GGG GGC 161 Ala Val Ala Ala Ala Val Gly Val Leu Leu Leu Ala Gly Ala Gly Gly 5 10 15 20 GCG GCA GGC GAC GAG GCC CGG GAG GCG GCG GCC GTG CGG GCG CTC GTG 209 Ala Ala Gly Asp Glu Ala Arg Glu Ala Ala Ala Val Arg Ala Leu Val 25 30 35 GCC CGG CTG CTG GGG CCA GGC CCC GCG GCC GAC TTC TCC GTG TCG GTG 257 Ala Arg Leu Leu Gly Pro Gly Pro Ala Ala Asp Phe Ser Val Ser Val 40 45 50 GAG CGC GCT CTG GCT GCC AAG CCG GGC TTG GAC ACC TAC AGC CTG GGC 305 Glu Arg Ala Leu Ala Ala Lys Pro Gly Leu Asp Thr Tyr Ser Leu Gly 55 60 65 GGC GGC GGC GCG GCG CGC GTG CGG GTG CGC GGC TCC ACG GGC GTG GCG 353 Gly Gly Gly Ala Ala Arg Val Arg Val Arg Gly Ser Thr Gly Val Ala 70 75 80 GCC GCC GCG GGG CTG CAC CGC TAC CTG CGC GAC TTC TGT GGC TGC CAC 401 Ala Ala Ala Gly Leu His Arg Tyr Leu Arg Asp Phe Cys Gly Cys His 85 90 95 100 GTG GCC TGG TCC GGC TCT CAG CTG CGC CTG CCG CGG CCA CTG CCA GCC 449 Val Ala Trp Ser Gly Ser Gln Leu Arg Leu Pro Arg Pro Leu Pro Ala 105 110 115 GTG CCG GGG GAG CTG ACC GAG GCC ACG CCC AAC AGG TAC CGC TAT TAC 497 Val Pro Gly Glu Leu Thr Glu Ala Thr Pro Asn Arg Tyr Arg Tyr Tyr 120 125 130 CAG AAT GTG TGC ACG CAA AGC TAC TCC TTC GTG TGG TGG GAC TGG GCC 545 Gln Asn Val Cys Thr Gln Ser Tyr Ser Phe Val Trp Trp Asp Trp Ala 135 140 145 CGC TGG GAG CGA GAG ATA GAC TGG ATG GCG CTG AAT GGC ATC AAC CTG 593 Arg Trp Glu Arg Glu Ile Asp Trp Met Ala Leu Asn Gly Ile Asn Leu 150 155 160 GCA CTG GCC TGG AGC GGC CAG GAG GCC ATC TGG CAG CGG GTG TAC CTG 641 Ala Leu Ala Trp Ser Gly Gln Glu Ala Ile Trp Gln Arg Val Tyr Leu 165 170 175 180 GCC TTG GGC CTG ACC CAG GCA GAG ATC AAT GAG TTC TTT ACT GGT CCT 689 Ala Leu Gly Leu Thr Gln Ala Glu Ile Asn Glu Phe Phe Thr Gly Pro 185 190 195 GCC TTC CTG GCC TGG GGG CGA ATG GGC AAC CTG CAC ACC TGG GAT GGC 737 Ala Phe Leu Ala Trp Gly Arg Met Gly Asn Leu His Thr Trp Asp Gly 200 205 210 CCC CTG CCC CCC TCC TGG CAC ATC AAG CAG CTT TAC CTG CAG CAC CGG 785 Pro Leu Pro Pro Ser Trp His Ile Lys Gln Leu Tyr Leu Gln His Arg 215 220 225 GTC CTG GAC CAG ATG CGC TCC TTC GGC ATG ACC CCA GTG CTG CCT GCA 833 Val Leu Asp Gln Met Arg Ser Phe Gly Met Thr Pro Val Leu Pro Ala 230 235 240 TTC GCG GGG CAT GTT CCC GAG GCT GTC ACC AGG GTG TTC CCT CAG GTC 881 Phe Ala Gly His Val Pro Glu Ala Val Thr Arg Val Phe Pro Gln Val 245 250 255 260 AAT GTC ACG AAG ATG GGC AGT TGG GGC CAC TTT AAC TGT TCC TAC TCC 929 Asn Val Thr Lys Met Gly Ser Trp Gly His Phe Asn Cys Ser Tyr Ser 265 270 275 TGC TCC TTC CTT CTG GCT CCG GAA GAC CCC ATA TTC CCC ATC ATC GGG 977 Cys Ser Phe Leu Leu Ala Pro Glu Asp Pro Ile Phe Pro Ile Ile Gly 280 285 290 AGC CTC TTC CTG CGA GAG CTG ATC AAA GAG TTT GGC ACA GAC CAC ATC 1025 Ser Leu Phe Leu Arg Glu Leu Ile Lys Glu Phe Gly Thr Asp His Ile 295 300 305 TAT GGG GCC GAC ACT TTC AAT GAG ATG CAG CCA CCT TCC TCA GAG CCC 1073 Tyr Gly Ala Asp Thr Phe Asn Glu Met Gln Pro Pro Ser Ser Glu Pro 310 315 320 TCC TAC CTT GCC GCA GCC ACC ACT GCC GTC TAT GAG GCC ATG ACT GCA 1121 Ser Tyr Leu Ala Ala Ala Thr Thr Ala Val Tyr Glu Ala Met Thr Ala 325 330 335 340 GTG GAT ACT GAG GCT GTG TGG CTG CTC CAA GGC TGG CTC TTC CAG CAC 1169 Val Asp Thr Glu Ala Val Trp Leu Leu Gln Gly Trp Leu Phe Gln His 345 350 355 CAG CCG CAG TTC TGG GGG CCC GCC CAG ATC AGG GCT GTG CTG GGA GCT 1217 Gln Pro Gln Phe Trp Gly Pro Ala Gln Ile Arg Ala Val Leu Gly Ala 360 365 370 GTG CCC CGT GGC CGC CTC CTG GTT CTG GAC CTG TTT GCT GAG AGC CAG 1265 Val Pro Arg Gly Arg Leu Leu Val Leu Asp Leu Phe Ala Glu Ser Gln 375 380 385 CCT GTG TAT ACC CGC ACT GCC TCC TTC CAG GGC CAG CCC TTC ATC TGG 1313 Pro Val Tyr Thr Arg Thr Ala Ser Phe Gln Gly Gln Pro Phe Ile Trp 390 395 400 TGC ATG CTG CAC AAC TTT GGG GGA AAC CAT GGT CTT TTT GGA GCC CTA 1361 Cys Met Leu His Asn Phe Gly Gly Asn His Gly Leu Phe Gly Ala Leu 405 410 415 420 GAG GCT GTG AAC GGA GGC CCA GAA GCT GCC CGC CTC TTC CCC AAC TCC 1409 Glu Ala Val Asn Gly Gly Pro Glu Ala Ala Arg Leu Phe Pro Asn Ser 425 430 435 ACC ATG GTA GGC ACG GGC ATG GCC CCC GAG GGC ATC AGC CAG AAC GAA 1457 Thr Met Val Gly Thr Gly Met Ala Pro Glu Gly Ile Ser Gln Asn Glu 440 445 450 GTG GTC TAT TCC CTC ATG GCT GAG CTG GGC TGG CGA AAG GAC CCA GTG 1505 Val Val Tyr Ser Leu Met Ala Glu Leu Gly Trp Arg Lys Asp Pro Val 455 460 465 CCA GAT TTG GCA GCC TGG GTG ACC AGC TTT GCC GCC CGG CGG TAT GGG 1553 Pro Asp Leu Ala Ala Trp Val Thr Ser Phe Ala Ala Arg Arg Tyr Gly 470 475 480 GTC TCC CAC CCG GAC GCA GGG GCA GCG TGG AGG CTA CTG CTC CGG AGT 1601 Val Ser His Pro Asp Ala Gly Ala Ala Trp Arg Leu Leu Leu Arg Ser 485 490 495 500 GTG TAC AAC TGC TCC GGG GAG GCC TGC AGG GGC CAC AAT CGT AGC CCG 1649 Val Tyr Asn Cys Ser Gly Glu Ala Cys Arg Gly His Asn Arg Ser Pro 505 510 515 CTG GTC AGG CGG CCG TCC CTA CAG ATG AAT ACC AGC ATC TGG TAC AAC 1697 Leu Val Arg Arg Pro Ser Leu Gln Met Asn Thr Ser Ile Trp Tyr Asn 520 525 530 CGA TCT GAT GTG TTT GAG GCC TGG CGG CTG CTG CTC ACA TCT GCT CCC 1745 Arg Ser Asp Val Phe Glu Ala Trp Arg Leu Leu Leu Thr Ser Ala Pro 535 540 545 TCC CTG GCC ACC AGC CCC GCC TTC CGC TAC GAC CTG CTG GAC CTC ACT 1793 Ser Leu Ala Thr Ser Pro Ala Phe Arg Tyr Asp Leu Leu Asp Leu Thr 550 555 560 CGG CAG GCA GTG CAG GAG CTG GTC AGC TTG TAC TAT GAG GAG GCA AGA 1841 Arg Gln Ala Val Gln Glu Leu Val Ser Leu Tyr Tyr Glu Glu Ala Arg 565 570 575 580 AGC GCC TAC CTG AGC AAG GAG CTG GCC TCC CTG TTG AGG GCT GGA GGC 1889 Ser Ala Tyr Leu Ser Lys Glu Leu Ala Ser Leu Leu Arg Ala Gly Gly 585 590 595 GTC CTG GCC TAT GAG CTG CTG CCG GCA CTG GAC GAG GTG CTG GCT AGT 1937 Val Leu Ala Tyr Glu Leu Leu Pro Ala Leu Asp Glu Val Leu Ala Ser 600 605 610 GAC AGC CGC TTC TTG CTG GGC AGC TGG CTA GAG CAG GCC CGA GCA GCG 1985 Asp Ser Arg Phe Leu Leu Gly Ser Trp Leu Glu Gln Ala Arg Ala Ala 615 620 625 GCA GTC AGT GAG GCC GAG GCC GAT TTC TAC GAG CAG AAC AGC CGC TAC 2033 Ala Val Ser Glu Ala Glu Ala Asp Phe Tyr Glu Gln Asn Ser Arg Tyr 630 635 640 CAG CTG ACC TTG TGG GGG CCA GAA GGC AAC ATC CTG GAC TAT GCC AAC 2081 Gln Leu Thr Leu Trp Gly Pro Glu Gly Asn Ile Leu Asp Tyr Ala Asn 645 650 655 660 AAG CAG CTG GCG GGG TTG GTG GCC AAC TAC TAC ACC CCT CGC TGG CGG 2129 Lys Gln Leu Ala Gly Leu Val Ala Asn Tyr Tyr Thr Pro Arg Trp Arg 665 670 675 CTT TTC CTG GAG GCG CTG GTT GAC AGT GTG GCC CAG GGC ATC CCT TTC 2177 Leu Phe Leu Glu Ala Leu Val Asp Ser Val Ala Gln Gly Ile Pro Phe 680 685 690 CAA CAG CAC CAG TTT GAC AAA AAT GTC TTC CAA CTG GAG CAG GCC TTC 2225 Gln Gln His Gln Phe Asp Lys Asn Val Phe Gln Leu Glu Gln Ala Phe 695 700 705 GTT CTC AGC AAG CAG AGG TAC CCC AGC CAG CCG CGA GGA GAC ACT GTG 2273 Val Leu Ser Lys Gln Arg Tyr Pro Ser Gln Pro Arg Gly Asp Thr Val 710 715 720 GAC CTG GCC AAG AAG ATC TTC CTC AAA TAT TAC CCC GGC TGG GTG GCC 2321 Asp Leu Ala Lys Lys Ile Phe Leu Lys Tyr Tyr Pro Gly Trp Val Ala 725 730 735 740 GGC TCT TGG TGATAGATTC GCCACCACTG GGCCTTGTTT TCCGCTAATT 2370 Gly Ser Trp CCAGGGCAGA TTCCAGGGCC CAGAGCTGGA CAGACATCAC AGGATAACCC AGGCCTGGGA 2430 GGAGGCCCCA CGGCCTGCTG GTGGGGTCTG ACCTGGGGGG ATTGGAGGGA AATGACCTGC 2490 CCTCCACCAC CACCCAAAGT GTGGGATTAA AGTACTGTTT TCTTTCCACT TAAAAAAAAA 2550 AAAAAAGTCG AGCGGCCGCG AATTC 2575 743 amino acids amino acid linear protein not provided Potentially-glycosylated Asn site, 261 Potentially-glycosylated Asn site, 272 Potentially-glycosylated Asn site, 435 Potentially-glycosylated Asn site, 503 Potentially-glycosylated Asn site, 513 Potentially-glycosylated Asn site, 526 Potentially-glycosylated Asn site, 532 2 Met Glu Ala Val Ala Val Ala Ala Ala Val Gly Val Leu Leu Leu Ala 1 5 10 15 Gly Ala Gly Gly Ala Ala Gly Asp Glu Ala Arg Glu Ala Ala Ala Val 20 25 30 Arg Ala Leu Val Ala Arg Leu Leu Gly Pro Gly Pro Ala Ala Asp Phe 35 40 45 Ser Val Ser Val Glu Arg Ala Leu Ala Ala Lys Pro Gly Leu Asp Thr 50 55 60 Tyr Ser Leu Gly Gly Gly Gly Ala Ala Arg Val Arg Val Arg Gly Ser 65 70 75 80 Thr Gly Val Ala Ala Ala Ala Gly Leu His Arg Tyr Leu Arg Asp Phe 85 90 95 Cys Gly Cys His Val Ala Trp Ser Gly Ser Gln Leu Arg Leu Pro Arg 100 105 110 Pro Leu Pro Ala Val Pro Gly Glu Leu Thr Glu Ala Thr Pro Asn Arg 115 120 125 Tyr Arg Tyr Tyr Gln Asn Val Cys Thr Gln Ser Tyr Ser Phe Val Trp 130 135 140 Trp Asp Trp Ala Arg Trp Glu Arg Glu Ile Asp Trp Met Ala Leu Asn 145 150 155 160 Gly Ile Asn Leu Ala Leu Ala Trp Ser Gly Gln Glu Ala Ile Trp Gln 165 170 175 Arg Val Tyr Leu Ala Leu Gly Leu Thr Gln Ala Glu Ile Asn Glu Phe 180 185 190 Phe Thr Gly Pro Ala Phe Leu Ala Trp Gly Arg Met Gly Asn Leu His 195 200 205 Thr Trp Asp Gly Pro Leu Pro Pro Ser Trp His Ile Lys Gln Leu Tyr 210 215 220 Leu Gln His Arg Val Leu Asp Gln Met Arg Ser Phe Gly Met Thr Pro 225 230 235 240 Val Leu Pro Ala Phe Ala Gly His Val Pro Glu Ala Val Thr Arg Val 245 250 255 Phe Pro Gln Val Asn Val Thr Lys Met Gly Ser Trp Gly His Phe Asn 260 265 270 Cys Ser Tyr Ser Cys Ser Phe Leu Leu Ala Pro Glu Asp Pro Ile Phe 275 280 285 Pro Ile Ile Gly Ser Leu Phe Leu Arg Glu Leu Ile Lys Glu Phe Gly 290 295 300 Thr Asp His Ile Tyr Gly Ala Asp Thr Phe Asn Glu Met Gln Pro Pro 305 310 315 320 Ser Ser Glu Pro Ser Tyr Leu Ala Ala Ala Thr Thr Ala Val Tyr Glu 325 330 335 Ala Met Thr Ala Val Asp Thr Glu Ala Val Trp Leu Leu Gln Gly Trp 340 345 350 Leu Phe Gln His Gln Pro Gln Phe Trp Gly Pro Ala Gln Ile Arg Ala 355 360 365 Val Leu Gly Ala Val Pro Arg Gly Arg Leu Leu Val Leu Asp Leu Phe 370 375 380 Ala Glu Ser Gln Pro Val Tyr Thr Arg Thr Ala Ser Phe Gln Gly Gln 385 390 395 400 Pro Phe Ile Trp Cys Met Leu His Asn Phe Gly Gly Asn His Gly Leu 405 410 415 Phe Gly Ala Leu Glu Ala Val Asn Gly Gly Pro Glu Ala Ala Arg Leu 420 425 430 Phe Pro Asn Ser Thr Met Val Gly Thr Gly Met Ala Pro Glu Gly Ile 435 440 445 Ser Gln Asn Glu Val Val Tyr Ser Leu Met Ala Glu Leu Gly Trp Arg 450 455 460 Lys Asp Pro Val Pro Asp Leu Ala Ala Trp Val Thr Ser Phe Ala Ala 465 470 475 480 Arg Arg Tyr Gly Val Ser His Pro Asp Ala Gly Ala Ala Trp Arg Leu 485 490 495 Leu Leu Arg Ser Val Tyr Asn Cys Ser Gly Glu Ala Cys Arg Gly His 500 505 510 Asn Arg Ser Pro Leu Val Arg Arg Pro Ser Leu Gln Met Asn Thr Ser 515 520 525 Ile Trp Tyr Asn Arg Ser Asp Val Phe Glu Ala Trp Arg Leu Leu Leu 530 535 540 Thr Ser Ala Pro Ser Leu Ala Thr Ser Pro Ala Phe Arg Tyr Asp Leu 545 550 555 560 Leu Asp Leu Thr Arg Gln Ala Val Gln Glu Leu Val Ser Leu Tyr Tyr 565 570 575 Glu Glu Ala Arg Ser Ala Tyr Leu Ser Lys Glu Leu Ala Ser Leu Leu 580 585 590 Arg Ala Gly Gly Val Leu Ala Tyr Glu Leu Leu Pro Ala Leu Asp Glu 595 600 605 Val Leu Ala Ser Asp Ser Arg Phe Leu Leu Gly Ser Trp Leu Glu Gln 610 615 620 Ala Arg Ala Ala Ala Val Ser Glu Ala Glu Ala Asp Phe Tyr Glu Gln 625 630 635 640 Asn Ser Arg Tyr Gln Leu Thr Leu Trp Gly Pro Glu Gly Asn Ile Leu 645 650 655 Asp Tyr Ala Asn Lys Gln Leu Ala Gly Leu Val Ala Asn Tyr Tyr Thr 660 665 670 Pro Arg Trp Arg Leu Phe Leu Glu Ala Leu Val Asp Ser Val Ala Gln 675 680 685 Gly Ile Pro Phe Gln Gln His Gln Phe Asp Lys Asn Val Phe Gln Leu 690 695 700 Glu Gln Ala Phe Val Leu Ser Lys Gln Arg Tyr Pro Ser Gln Pro Arg 705 710 715 720 Gly Asp Thr Val Asp Leu Ala Lys Lys Ile Phe Leu Lys Tyr Tyr Pro 725 730 735 Gly Trp Val Ala Gly Ser Trp 740 10380 base pairs nucleic acid single linear DNA (genomic) Homo sapiens Chromosome 17 exon 1 990..1372 exon 2 2115..2262 exon 3 3056..3202 exon 4 3387..3472 exon 5 5667..5923 exon 6 7745..8955 3 ATAATGAGCA GTGAGGACGA TCAGAGGTCA CCTTCCTGTC TTGGTTTTGG CAGGTTTTGA 60 CCAGTTTCTT TGCTGCATTC TGTTTTATCA GCGGGGTCTT GTGACCTTTT ATCTTGTGCT 120 GACCTCCTGT CTCATCCTGT GACGAAGGCC TAACCTCCTG GGAATTCAGC CCAGCAGGTC 180 TCTGCCTCAT TTTACCCAGC CCCTGTTCAA GATGGAGTCG CTCTGGTTGG AAACTTCTGA 240 CAAAATGACA GCTCCTGTTA TGTTGCTGCT GCTGCCGCCA ATGGACAGCC TTTAACGTGC 300 CCGCCAGCCC TGCTCCACCG CCGGCCTGGG CTCACATGGC CCCATCCCTC CTCGAACCTC 360 CTAGCCTGTT AGTTACTCAA ATCTGCAAGC TCTCTGCCTT CTCAGGGCCT TCAATAAATG 420 CATTTCTTCT GTCTGGAAGG CTCTTCCTTT CCCTCTTCTA GCCAATTCCT ATTCATCCCT 480 GAGTTTCAGA TTAAAAGTCA CTTCCTTTGG AAACCTTACT TCGCTACTTC GCTACTTACT 540 GCACTACTTC GCAGCATCAC AACTATGATG GAAATCCTTA CTTACGTTAA ATATCTGGTT 600 TCTAGGTCAC CTCCCTGACG GGGACGGTAG GGACCGTCTT CTCGTTCATC AGTAGGGAAG 660 TAGCTATGGC AGTGCCTGAT ACAAAATAAA CTCCAAATGT GTATTTATTA GATGGTTGGA 720 TGGAAGTTAT TTGCGTGTGA AAGCGCGTTT TACCCGAAGG CGCTCTGTGA GGGCCAGCGG 780 GTCCCCTTCG GCCCTGGAGC CGGGGTCACA CGCTCCCCAC CGCGTGCGGT CACGAGACGC 840 CCCCAAGGGA GTATCCTGGT ACCCGGAAGC CGCGACTCCT GGCCCTGAGC CCGGGCTTAG 900 CCTTCGGGTC CACGTGGCCG GAGCCGGCAG CTGATTGGAC GCGGGCCGCC CCACCCCCTG 960 GCCGTCGCGG GACCCGCAGG ACTGAGACCA TGGAGGCGGT GGCGGTGGCC GCGGCGGTGG 1020 GGGTCCTTCT CCTGGCCGGG GCCGGGGGCG CGGCAGGCGA CGAGGCCCGG GAGGCGGCGG 1080 CCGTGCGGGC GCTCGTGGCC CGGCTGCTGG GGCCAGGCCC CGCGGCCGAC TTCTCCGTGT 1140 CGGTGGAGCG CGCTCTGGCT GCCAAGCCGG GCTTGGACAC CTACAGCCTG GGCGGCGGCG 1200 GCGCGGCGCG CGTGCGGGTG CGCGGCTCCA CGGGCGTGGC GGCCGCCGCG GGGCTGCACC 1260 GCTACCTGCG CGACTTCTGT GGCTGCCACG TGGCCTGGTC CGGCTCTCAG CTGCGCCTGC 1320 CGCGGCCACT GCCAGCCGTG CCGGGGGAGC TGACCGAGGC CACGCCCAAC AGGTACCGCC 1380 CCGAAGCTTC CCCGCGTCCG CCCGAGGCGC TTACCCCCTC CCGGAGCCGC TGCCACCCAA 1440 ATCGGGAGGC TGAGCGGGGA GCGCTGGCCG GAAGGCCCAG CTGCGCCGCC TCCAGCAGCT 1500 GTGTGGCCTT GAGCCAGCCA CTCTGCCTTT CAGAGCCTCG GCTGGCCCAC CTGAAAAACG 1560 GAAAGAAGAC GCCTACCGTG CAGTGTTATT GTGAGGATTT GCACGATGAT GGGCATAGAA 1620 TTTGTGGTGC ACAATTGGTG ATGAGTGAAT TTTCTTGCCT TCCTCCCCCA CCTTCTCTTT 1680 GAACCTGCGG ACTGAGGAAG GACGCCTCCA TCCCCCACCC TACAGGCCTG TGTTCCAGCG 1740 CCTGCCACAC TATGGAGTGA TGTGTTCACA CAGCTGTCCT CCCCTGCCCA TCTGTTAGAC 1800 TGTGGGGGCA GGGATTCCCC GTTCCAGGAA AACACCGTGC AGAGGAGGGG CTCTGGCAGT 1860 GTGGCATGAA AGTGGAATAT GCCACCCAAA TACCCGCCAG GCTAGAGGGC CCTGGGAGAG 1920 TGCAGGGGAC GAGTGCCTCA GAAGCCCAGC CCCGGTACCT GGTCTCAGCT CCACCTGGGG 1980 TGGGTCCCAG TGTGCAGCAG AAGGGCCGAG TTTGGAGCCC CTCCCCTCTC CTCTAGGTGG 2040 GGGATGGGGG ATTTGTTCCA GGGCCGTGGA CCCTCCAGGG TGGGATGCGC CCCTGCTCAT 2100 GACACTGCCC GCAGGTACCG CTATTACCAG AATGTGTGCA CGCAAAGCTA CTCCTTCGTG 2160 TGGTGGGACT GGGCCCGCTG GGAGCGAGAG ATAGACTGGA TGGCGCTGAA TGGCATCAAC 2220 CTGGCACTGG CCTGGAGCGG CCAGGAGGCC ATCTGGCAGC GGGTGCGTGC CCACTGTCCC 2280 TTCCCCACCC TCCTCTATGG CGGGAGCCAC CGTAGGTGTT TTCACCCGCC CCCCAGCATG 2340 GGCGCAGTGT CTCTCTCTAG AAGTGCTTTC AGCGTGCACA GTGGCTTGGG CCTCCTAAAA 2400 ACTGAGGCTT CCGGCCGGGC GCGGTGGCTC ACGCCTGTCA TCCCAGCACT TCGGGAGGCC 2460 TAGGCGGGCG GATCAGGAGT TCAGGAGATC GAGACCATCC TGGCCAACAT TGTGAAACCC 2520 CGTCTCTACT AAAATACAAA GAAATAGCAA CCTGGGCAAC AGAGCGAGAC TCTGTCTAAA 2580 AAAAAAAAAA AAAAAAACTG AGGCTTCCAG TTTGAGGAGT GGGGCTCCTT CCCCCATCTC 2640 CCCTATGCAG CCAATCACCT GGTCCCTTGG ATCCAACTCA TGGGCAGCTC TAGATCTGCC 2700 TCCCTGGAAG CTTCTGTGCT GCAATGGCTG CTCCAGGCTC TGCTTAAGCT CTTCACACAG 2760 TTGCCCTGCC CTTCCATCTG GCACTCTTGC TCCATGAAGC CTTCTAAGGC CTTCCTGTTG 2820 GGGGAAAGCC CCTTTGTGCC CCATCTCCTC ACCCATGCGA CAAAGGCAAC ACAGTGAACT 2880 CACCTACTCA CAGGTCTCTT TCCTCTGGGC TGTGGGCTCC TTGATGGCAG CGTTCGGATT 2940 TTGTCTCAGT AGCCCTAGCA CCCAGCACAA AGAAGCAATG AGTGAATGGT TGTTGAATGA 3000 ATGAATGAAT GAATGAAGAT GAATATATTT CTATGTGTGG GCCCTTCTTC CTCAGGTGTA 3060 CCTGGCCTTG GGCCTGACCC AGGCAGAGAT CAATGAGTTC TTTACTGGTC CTGCCTTCCT 3120 GGCCTGGGGG CGAATGGGCA ACCTGCACAC CTGGGATGGC CCCCTGCCCC CCTCCTGGCA 3180 CATCAAGCAG CTTTACCTGC AGGTAAAAGG ATGGAAAAGG GAAGGGGCAG AATCGGTGAT 3240 AGATGGTCAT GGGCCCAGGA AGGGTGGTAT TAGGCCGGCC CCAGGGCTCT TAACTGAGGC 3300 GGGGGGCTGC GTGTATCCTG GGAGATGAGG GCCTTCTCAT AGGACAGCAG TGGCCATGCT 3360 CACCACCCTT CCTTCTGTTC CTCCAGCACC GGGTCCTGGA CCAGATGCGC TCCTTCGGCA 3420 TGACCCCAGT GCTGCCTGCA TTCGCGGGGC ATGTTCCCGA GGCTGTCACC AGGTGAGGTT 3480 CCGCTCACCC CCTCCACTTA GCTCAGAGAG GGAATTTTAT TCCCTTCTAG AACATGACTT 3540 AAAAACTTAA GCTCTGGGCC GGGCGCAGTG GCTCACGCCT GTAATCCCAG CACTTTGGGA 3600 GGCCGAGTTG GGCGGATCAC CTGAGGTCAG GAGTTCGAGA CCAGCCTGGC CAACATGGTG 3660 AAACCCTGTC TCTACTAAAA ATATAAAAAT TAGCTGGGCA TGGTGGCACG CGCCTGTAAT 3720 CCCATCTACT TAGGAGGCTG AGACAGGAGA ATTGCTTAAA CCTGGGAGGC AGACGTTGCA 3780 GTGAGTCAAG ATCACGCCAT TGCACTCCAG CCTGGGTGAC GAGCGAAACT CTGTCTCAAA 3840 CAAACAAACA AGCTCTGGAC GTAGGCCTGG GTTTGATTTC TGACTCTGCT ACTAATTAGC 3900 TGTGTGACTT CGGGCAGATG ACATGACTGC TCTGTGCCTC AGTTTCCTTA CTTGTAAAAT 3960 GGGATCTCTA CCCACTTCGC TGTAGGGTTT GTAATTATCT CTCGATCTAT CTGTGACTTT 4020 GCACAGAGTG CTAGCAAATG GCAGCCCTTG GGAGTGGCAG CAGGGGTGCT CCAGTGTCCC 4080 TTGTCCCTCC TGTTCCTCTG TGCTTCCCAG CCATCCTCTC ACATGTGGTT GGGAAAAGTC 4140 TTCAAGGCTC ACCTGAGACC TCCCCTCCTT CAGGAAGCCT TGCTAGTGCC CCGCATGACC 4200 TCCTTTGCAC CTGCTAATGT CTGGCTCCCA TACTCTCGTA GGACTTAATG CATGCCAGTG 4260 GCCTCCCTGC CCGCCTCTTT GCCCCCATCA CCAGGTGGCA GGAAACTCAC TCATTCATTC 4320 AATAAACTTG GTCCAGCTGT CTGAGGCTGC CAGAACTGGC TGTGCTGGGT CCTGGGAGGC 4380 GGCAAGAAAG GTGCCCAAGG GCTTACCCCT GATAGGAGAG ATATGTTGGC TGAAGGATAC 4440 AATGTGGGGA CAAGGACAGG AATATATGTG GGTTCCGCTC TCCTCTGCCG GGAGAGAGGG 4500 GCAGGAAGGG CTCAGGGCAG AGCCCAGCCT TGAAAAATGA GTGTTGCTTG GACGGACGCT 4560 TGGCTAATGC TTGTAATCCT AGCGTTTTGG GAGGCTGAGG CGTATGGATC ACCTGCGGTC 4620 AGGAGTTAAA GACCAGCCTG GCCAACATGG CGAAACCCCA TCTCTACTAA AAGTACAAAA 4680 ATTAGCCAGG CGTGGTGGCG GGCTCCTGTA ATCCCAGCTA CTCGGTAGGC TGAGGCATGA 4740 GAATCTCTTG AAGCCAGGGG CCAGAGACTG CAGTGAGCCG AGATCACACC ACTTCACTCC 4800 AGCCTGGGTG ACAGAGTGAG ACTCCGTCTC AAAAAAAAAA AAAAAAAAAG GAAAGAAAAT 4860 TAAACACCTC ATGTTCTCAC TCATAGTGGG AGTTGAACAA TGAGAACAAC ATGGACACAG 4920 GAAGGGGAAC ATCACACACC GGGGCCTTTC GCGGTGTGGG GGTCAAGGGG AGGAGTAGCA 4980 TTGGGACAGA TACTTAATGC ATGCGGGGCT GAAAACCTAG ATGATGGGTT GATGGGTGCA 5040 GCAAACCACC ATGGCACATG TATACCTATG CAACAAACCT GCATGTTCTG CACAGAACTG 5100 AACTGAAAGT ATAATTAAAA AAAAAAAAAA AAGCTGGGTG CGGTGGCCCA CACCTGTAAT 5160 CCCAGCACTT TGGGAGGCCG AGACGGGCGG ATCACAAGGT CAGCAGATCG AGACCATCCT 5220 GGCTAACACA GTGAAACTCA GTCTCTACTA AAAATACAAA AAATTAGCCG GGTGTGGTGG 5280 CGGGCACCTG TAGTCCCAGC TACTAGGGAG GCTGAGGCAG GAGAATGGCA TGAACCTGGG 5340 AGGCAGAGCT TGCAGTGAGC TGAGAATGCG CCACTGCACT CCAGCCTGGG GGACAGAGTG 5400 AGACTCTGCC TCAAAAAAAA AAAAAAAAAG AAAGAAAAAG GAGCGTTGCT TGTTTCAGGC 5460 CACAGGAAGG GGAGAGATAG TGAAAGTTTT TCAGAGAAGG TGGCCAGGGA AGGAGAAGAA 5520 AGGACTGTAG GCAGAGAGCA TAGCCTGTAC AAAGCCATAG AGGCAAGAGA AACCAGGAGC 5580 TGTAGAGAAG TTGGCAAGGC TGTTGAACAC TATGGTGAAC ACTATGGCGG CTTCCATGAA 5640 ATATCTGAGC TTTTGCTCCC CACTAGGGTG TTCCCTCAGG TCAATGTCAC GAAGATGGGC 5700 AGTTGGGGCC ACTTTAACTG TTCCTACTCC TGCTCCTTCC TTCTGGCTCC GGAAGACCCC 5760 ATATTCCCCA TCATCGGGAG CCTCTTCCTG CGAGAGCTGA TCAAAGAGTT TGGCACAGAC 5820 CACATCTATG GGGCCGACAC TTTCAATGAG ATGCAGCCAC CTTCCTCAGA GCCCTCCTAC 5880 CTTGCCGCAG CCACCACTGC CGTCTATGAG GCCATGACTG CAGGTACAGT GCCTGGGTGG 5940 GGTGGGAGAG CCCCCCAGAC CCTCAAAAAG AAGGGAGTAG CAGATGTCAG TAGGGGTAGG 6000 CAGAGGGACT GGAATAATGC CTCGCCATAA CACACAGTAC TTTATAGTTT ACCAAGCACG 6060 TGTACACATG CGTTGTCTCA GTGAATCCCA CTGTGGTTGA GAGGTGAGCT CTGGAAGCCA 6120 ACAACCTGGG TCACACCTCG CGCTCCTATT TCCTGGCCGT GTGACTTATG ACTCATGACC 6180 TCCTTCCCAG TGTCTCGTTT GCTTTTCCTG TAAACTGGGA CTACCTCATA GGTAGAATAA 6240 CGCCTGGCCC AGAGCAAAGG CCACTAAGAG CTAGCTATGA ACAAGGATTT TGTTTCATCT 6300 CTGCGTGGTT GCTGAAGTAG GCACTGCAGG CAGGAGGTGA GTGGATGTGC CTAAAGGCAC 6360 TAAGTGCGCA TCCTGCTACA AAACTGTGAA GCCAGGGCTC CTTCCTGCCA CTTAAAGGAG 6420 GAGTGGAGCA GAGGGCGCCC AAGTCAGGAA TGACTTAGTG GAGAGGCGTC TGTGTTGGCC 6480 AGGAAGGGAA CAGATCAGCT CAGCCTTTCT TGAGCAGTAC TGCTCCAAGT GTGACCCAAA 6540 ACCAGCAGCA GCAGCAGCAG CAGCCCGAGC TGTGAGATGG CAAATTCTCA GGCCCTACCC 6600 AAGACCTGAA GGAGAAGCTA CATTTTTTTT TTTTTTGAGA CAGATTTCAC TCTGTTGCTG 6660 AGGCTGGAGC ACAGTGGCAC AATCTCATCT CACTGCAACC TTCGTCTCCT AGGTTCAAGC 6720 GATTCTCCTG CCTCAGCCTC CCGAGTAGCT GGGACTATAG GCACCCGCCA CCACGCCCGG 6780 CAATTTTTGT TTGTTTTGAG ATAGAGTCTC GCTCTGTCAC CCAGGCTGGA GTGCAGTGGC 6840 ACGATCTCAG TTCACTGCAA CCTCTGCTTC CTGAGTTCAA GCGATTCTCC TGCCTCAGCC 6900 TCCTGAGTAG CTGGGATTAC AGGCGCCCCC CAACCACACT CGGCTAATTT TTGTATTTTT 6960 AGTAGAGACG GGGTTTCGCT ATGTAGGTCA AGCTGGTTTC AAACTCCTGA CCTCAAATGA 7020 TTCGCCCACT TCAGCCTCCC AAAGTGCTGG GATTACAGGT GTGAGCCACC TTGCCTGGCC 7080 AATTTTTGTA TTTTTAGTAG AAACAGGTTT CACCATGGTG GCCAGACTGG TCTCAAACTC 7140 CTGACCTCAG GTGAACTGCC CACCTCAGCC TCCCAAAGTA CTGGTATTAC AGGCGTGATC 7200 CACTGCGACT GGCCTTGATT TTGTTTTTGA GACAGAATCT TACTCTGTCG CCCAGACTGG 7260 AGTGCAGTGG CACAATCTCA GCTCACTGCA ACTTCTGCCT CATGGGTTCA AGTGATTCTT 7320 GTGCCTCTAC CTCCCGAGTA GCCGGGATTA CAGGCACCTG CCATTACGCT AGGCTAATTT 7380 TTGTATTTTT AGTATAGACA GGGTTTCCCC ACATTGGCCA GGCTGGTCTG GAACTCCTGG 7440 GCTCAAGTGA TCCACCTGCT TCAGCCCCTC AGAGTACTGG GATTATAGGT GTGGGCCACC 7500 ACGCCCATTC AGAAACCTCC ATGTTTTAAG GAGCCCTCTG GGTAACTCTC ATGTTCACCC 7560 AAGCTGCTGA ACCCTGTCCT GGAGTTTTCA GAGGGACGCG TATGTGCCAC AGAGCGTCCC 7620 GCTGGTGGGG GTCATGGGAA GCCATGACCT GGGATAGACA GTCGTCTGTA GAGTGGGGTG 7680 AACATTCCCT GGGCCCTCTG TTTCATCACT CCTCTTCTCT GTTCCCCCTA CCTCCTGTCC 7740 ACAGTGGATA CTGAGGCTGT GTGGCTGCTC CAAGGCTGGC TCTTCCAGCA CCAGCCGCAG 7800 TTCTGGGGGC CCGCCCAGAT CAGGGCTGTG CTGGGAGCTG TGCCCCGTGG CCGCCTCCTG 7860 GTTCTGGACC TGTTTGCTGA GAGCCAGCCT GTGTATACCC GCACTGCCTC CTTCCAGGGC 7920 CAGCCCTTCA TCTGGTGCAT GCTGCACAAC TTTGGGGGAA ACCATGGTCT TTTTGGAGCC 7980 CTAGAGGCTG TGAACGGAGG CCCAGAAGCT GCCCGCCTCT TCCCCAACTC CACCATGGTA 8040 GGCACGGGCA TGGCCCCCGA GGGCATCAGC CAGAACGAAG TGGTCTATTC CCTCATGGCT 8100 GAGCTGGGCT GGCGAAAGGA CCCAGTGCCA GATTTGGCAG CCTGGGTGAC CAGCTTTGCC 8160 GCCCGGCGGT ATGGGGTCTC CCACCCGGAC GCAGGGGCAG CGTGGAGGCT ACTGCTCCGG 8220 AGTGTGTACA ACTGCTCCGG GGAGGCCTGC AGGGGCCACA ATCGTAGCCC GCTGGTCAGG 8280 CGGCCGTCCC TACAGATGAA TACCAGCATC TGGTACAACC GATCTGATGT GTTTGAGGCC 8340 TGGCGGCTGC TGCTCACATC TGCTCCCTCC CTGGCCACCA GCCCCGCCTT CCGCTACGAC 8400 CTGCTGGACC TCACTCGGCA GGCAGTGCAG GAGCTGGTCA GCTTGTACTA TGAGGAGGCA 8460 AGAAGCGCCT ACCTGAGCAA GGAGCTGGCC TCCCTGTTGA GGGCTGGAGG CGTCCTGGCC 8520 TATGAGCTGC TGCCGGCACT GGACGAGGTG CTGGCTAGTG ACAGCCGCTT CTTGCTGGGC 8580 AGCTGGCTAG AGCAGGCCCG AGCAGCGGCA GTCAGTGAGG CCGAGGCCGA TTTCTACGAG 8640 CAGAACAGCC GCTACCAGCT GACCTTGTGG GGGCCAGAAG GCAACATCCT GGACTATGCC 8700 AACAAGCAGC TGGCGGGGTT GGTGGCCAAC TACTACACCC CTCGCTGGCG GCTTTTCCTG 8760 GAGGCGCTGG TTGACAGTGT GGCCCAGGGC ATCCCTTTCC AACAGCACCA GTTTGACAAA 8820 AATGTCTTCC AACTGGAGCA GGCCTTCGTT CTCAGCAAGC AGAGGTACCC CAGCCAGCCG 8880 CGAGGAGACA CTGTGGACCT GGCCAAGAAG ATCTTCCTCA AATATTACCC CGGCTGGGTG 8940 GCCGGCTCTT GGTGATAGAT TCGCCACCAC TGGGCCTTGT TTTCCGCTAA TTCCAGGGCA 9000 GATTCCAGGG CCCAGAGCTG GACAGACATC ACAGGATAAC CCAGGCCTGG GAGGAGGCCC 9060 CACGGCCTGC TGGTGGGGTC TGACCTGGGG GGATTGGAGG GAAATGACCT GCCCTCCACC 9120 ACCACCCAAA GTGTGGGATT AAAGTACTGT TTTCTTTCCA CTTAAACTGA TGAGTCCCCT 9180 GGGTCTGTCA AAATGAGAAG GTCACTGCTG CCACGCTTGG GAGGACTCAG GGCTATAGCA 9240 TGGCCCTGGG GTGGGACCTG TTCTCCCATC CCTTGCCTCA CGTCCCTGTT TTTGTTTGTT 9300 TGTTTGTTTG TGACGGAGCC TTGGTCTGTT GCCCAGGCTT GAGTACAATG GCACAGTCTC 9360 GGCTCACTGC AACCTCCGCC TCCTGGGTTC AAGCAATTCT TGTGCCTCAG CCTCCCCGGT 9420 AGCTGGGACT ATAGGCATGC ACCACCACAC CAGGCTAATT TTTTTTTTTC CAAGATGGAG 9480 TCTTGCTCTG TCGCCCAGGT TGGAGTTTAG TGGCACCATA TTGGTTTACT GCAACCTCTG 9540 CCTCCCGGGT TCAAGCAATT CTCCTGCCTC AGTCTACCAG GGAGTTAGGA CTACGGGCCT 9600 GTGCCATCAC GCCTGGCTAA TTTTTGTATT TTTCATAGAG ATAAGGTTTC ACCATGTTGG 9660 CCAGGCTGGT CTTTAACTCC TGAACTCAAG TGATCCACCT GCCTCGGCCT TCCAAAGTGC 9720 TGGGATTACA GGAGTGAGCC ACCGTGCCCG GCCATGTCTC TCTTTTTAAC ACTAATGTTA 9780 CCCTGACCTT TGAACGTAGA ATGCCCTTCT GTTGCAGGAA AACCTCTTTT CAAACCATGT 9840 TTGTCCTTTG CTGGCATGCC ACAGCAACAG TCACCAACAC AGAAGACTTC TGTGACCAAA 9900 TATTTGGAGG ATTTTCCCCA CACACACCAA GCAGCAGACA TCAGCTGGGT GTCCTCCAAT 9960 TCAGTTCCAA TGTAATCAAC CAGAGACAGC ATCAGATCCC ACAGGGTTAG GGTGCAGATC 10020 CATGAGACCA CCCCCTCCTT CCCAACGGTT ACAAGTCCTG ATCCCTGGAA CTTCTGACTA 10080 ACTGGCTTCA AGTTGGAGTT CCCATGACCC CCTTCCCCTC TTTGGAGTCA ACTCATTTGC 10140 GACAGTGACC CACGAAACAC AGGGAAACCC TTATTATGTT TATTGCTTTA TTACAGAGGA 10200 AAAAAATTTT TTTCTTTCTT TTTTGAGACA GGGTCTCACT CTGTCATCCA GAATGACTGC 10260 AGTGGCAGGA TCTGGCTCCG TCACCCAGGC TGGAGTGCAG TGGCATGATC TCGGCTCACT 10320 ACAGCCTCCA TCCCCCCCAA ACCCCACGCC TCAGCGCCCC ACCCCGCAAG TGGCTGGGAC 10380 20 amino acids amino acid linear peptide NO N-terminal Homo sapiens Modified-site 10 4 Asp Glu Ala Arg Glu Ala Ala Ala Val Arg Ala Leu Val Ala Arg 1 5 10 15 Leu Leu Gly Pro Gly 20 18 amino acids amino acid linear peptide NO N-terminal Homo sapiens Modified-site, glycosylated or phosphorylated, wherein Xaa may be any amino acid residue, preferably Arg. 16 5 Lys Pro Gly Leu Asp Thr Tyr Ser Leu Gly Gly Gly Gly Ala Ala Xaa Val 1 5 10 15 Arg 15 amino acids amino acid linear peptide NO internal Homo sapiens Modified-site, glycosylated or phosphorylated, wherein Xaa may be any amino acid residue, preferably Ala 12 Modified-site, glycosylated or phosphorylated, wherein Xaa may be any amino acid residue, preferably Ser 14 6 Trp Arg Leu Leu Leu Thr Ser Ala Pro Ser Leu Xaa Thr Xaa Pro 1 5 10 15 

What is claimed is:
 1. An isolated nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence of nucleotides which encodes a human α-N-acetylglucosaminidase having the amino acid sequence set forth in SEQ ID NO:2 or an enzymatically active fragment thereof.
 2. The isolated nucleic acid molecule of claim 1 wherein the nucleotides are deoxyribonucleotides.
 3. The isolated nucleic acid molecule of claim 2 wherein said molecule is a cDNA.
 4. The isolated nucleic acid molecule of claim 2 wherein said molecule is a genomic DNA molecule.
 5. The isolated nucleic acid molecule of claim 1 isolated from liver, kidney or placenta.
 6. The isolated nucleic acid molecule of claim 1 having a nucleotide sequence as set forth in SEQ ID NO:1 or complementary thereto.
 7. The isolated nucleic acid molecule of claim 1 having a nucleotide sequence as set forth in SEQ ID NO:3 or complementary thereto.
 8. A genetic construct capable of replication in a eukaryotic cell or prokaryotic cell, said genetic construct comprising an isolated nucleic acid molecule which comprises a sequence of nucleotides which encodes or is complementary to a sequence which encodes a human α-N-acetylglucosaminidase having the amino acid sequence set forth in SEQ ID NO:2 or an enzymatically active fragment thereof.
 9. The genetic construct of claim 8 wherein the isolated nucleic acid molecule is under the control of a promoter sequence capable of regulating expression of the nucleic acid molecule.
 10. The genetic construct of claim 9 wherein the isolated nucleic acid molecule is capable of being expressed in cells derived from a eukaryote.
 11. The genetic construct of claim 10 wherein the isolated nucleic acid molecule is further capable of being expressed in cells derived from a mammal.
 12. The genetic construct claim 11 wherein the isolated nucleic acid molecule is further capable of being expressed in CHO cells.
 13. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide capable of hydrolysing the terminal α-N-acetylglucosamine residues present at the non-reducing terminus of fragments of heparan sulphate and heparin and wherein said nucleotide sequence is capable of hybridising under high stringency conditions to the nucleotide sequence set forth in SEQ ID NO:
 1. 14. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide capable of hydrolysing the terminal α-N-acetylglucosamine residues present at the non-reducing terminus of fragments of heparan sulphate and heparin and wherein said nucleotide sequence is capable of hybridising under high stringency conditions to the nucleotide sequence set forth in SEQ ID NO:3.
 15. A genetic construct comprising the nucleic acid molecule according to claim 1 under the control of a promoter sequence capable of regulating expression of the nucleic acid molecule in a eukaryotic or prokaryotic cell to produce a recombinant human α-N-acetylglucosaminidase.
 16. The genetic construct of claim 15 wherein the promoter is capable of regulating expression of the recombinant α-N-acetylglucosaminidase in a mammalian cell.
 17. The genetic construct of claim 16 wherein the promoter is the CMV promoter sequence or a promoter derived therefrom.
 18. The genetic construct of claim 15 further comprising a transcription terminator sequence.
 19. A genetic construct comprising the nucleic acid molecule according to claim 13 under the control of a promoter sequence capable of regulating expression of the nucleic acid molecule in a eukaryotic or prokaryotic cell to produce a recombinant human α-N-acetylglucosaminidase.
 20. A genetic construct comprising the nucleic acid molecule according to claim 14 under the control of a promoter sequence capable of regulating expression of the nucleic acid molecule in a eukaryotic or prokaryotic cell to produce a recombinant human α-N-acetylglucosaminidase.
 21. A genetic construct comprising the nucleic acid molecule according to claim 6 under the control of a promoter sequence capable of regulating expression of the nucleic acid molecule in a eukaryotic or prokaryotic cell to produce a recombinant human α-N-acetylglucosaminidase.
 22. A genetic construct comprising the nucleic acid molecule according to claim 7 under the control of a promoter sequence capable of regulating expression of the nucleic acid molecule in a eukaryotic or prokaryotic cell to produce a recombinant human α-N-acetylglucosaminidase.
 23. A eukaryotic cell comprising a recombinant human α-N-acetylglucosaminidase having the amino acid sequence set forth in SEQ ID No:
 2. 24. A eukaryotic cell which comprises the genetic construct of claim 8, wherein the isolated nucleic acid molecule is under the control of a promoter sequence capable of regulating expression of the nucleic acid molecule in the eukaryotic cell.
 25. A eukaryotic cell which comprises the genetic construct of claim 19, wherein the isolated nucleic acid molecule is under the control of a promoter sequence capable of regulating expression of the nucleic acid molecule in the eukaryotic cell.
 26. A eukaryotic cell which comprises the genetic construct of claim 20, wherein the isolated nucleic acid molecule is under the control of a promoter sequence capable of regulating expression of the nucleic acid molecule in the eukaryotic cell.
 27. A eukaryotic cell which comprises the genetic construct of claim 21, wherein the isolated nucleic acid molecule is under the control of a promoter sequence capable of regulating expression of the nucleic acid molecule in the eukaryotic cell.
 28. A eukaryotic cell which comprises the genetic construct of claim 22, wherein the isolated nucleic acid molecule is under the control of a promoter sequence capable of regulating expression of the nucleic acid molecule in the eukaryotic cell.
 29. The eukaryotic cell of claim 24 wherein the eukaryotic cell is a mammalian, yeast, or insect cell.
 30. The eukaryotic cell of claim 25 wherein the eukaryotic cell is a mammalian, yeast, or insect cell.
 31. The eukaryotic cell of claim 26 wherein the eukaryotic cell is a mammalian, yeast, or insect cell.
 32. The eukaryotic cell of claim 27 wherein the eukaryotic cell is a mammalian, yeast, or insect cell.
 33. The eukaryotic cell of claim 28 wherein the eukaryotic cell is a mammalian, yeast, or insect cell.
 34. The eukaryotic cell of any of claims 24, 32, or 33, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.
 35. The eukaryotic cell of claim 30 wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.
 36. The eukaryotic cell of claim 31 wherein the mammalian cell is a Chinese hamster ovary (CHO) cell. 